anti bai1 g8 igm mouse mab  (R&D Systems)

Journal: Investigative Ophthalmology & Visual Science

Article Title: Myo/Nog Cells Give Rise to Myofibroblasts During Epiretinal Membrane Formation in a Mouse Model of Proliferative Vitreoretinopathy

doi: 10.1167/iovs.64.2.1

Figure Lengend Snippet: Number of Cells Labeled With Antibodies to Markers of Myo/Nog Cells and Muscle in ERMs and the Retina

Article Snippet: Myo/Nog cells were identified in tissue sections by double labeling with the anti-BAI1 G8 IgM mouse mAb and the anti-noggin goat polyclonal anti-serum (AF719; R&D Systems, Minneapolis, MN, USA), as described previously., Double labeling also was carried out with the IgM BAI1 mAb and IgG mAb antibodies to α-SMA (F3777 diluted 1:250; Sigma-Aldrich, St. Louis, MO, USA), striated muscle myosin II (ab58899 diluted 1:200; Abcam, Cambridge, MA, USA), the leukocyte markers CD68 (ab201340 diluted 1:200; Abcam), CD45 (ab10558 diluted 1:200; Abcam), and CD18 (MA1819 diluted 1:100; ThermoFisher Scientific, Waltham, PA, USA), and human nucleoli (ab190710 diluted 1:100; Abcam).

Techniques: Labeling

Journal: Investigative Ophthalmology & Visual Science

Article Title: Myo/Nog Cells Give Rise to Myofibroblasts During Epiretinal Membrane Formation in a Mouse Model of Proliferative Vitreoretinopathy

doi: 10.1167/iovs.64.2.1

Figure Lengend Snippet: Number of Cells Labeled With Antibodies to Markers of Myo/Nog Cells and Muscle in the Ciliary Body, Lens, and Cornea

Article Snippet: Myo/Nog cells were identified in tissue sections by double labeling with the anti-BAI1 G8 IgM mouse mAb and the anti-noggin goat polyclonal anti-serum (AF719; R&D Systems, Minneapolis, MN, USA), as described previously., Double labeling also was carried out with the IgM BAI1 mAb and IgG mAb antibodies to α-SMA (F3777 diluted 1:250; Sigma-Aldrich, St. Louis, MO, USA), striated muscle myosin II (ab58899 diluted 1:200; Abcam, Cambridge, MA, USA), the leukocyte markers CD68 (ab201340 diluted 1:200; Abcam), CD45 (ab10558 diluted 1:200; Abcam), and CD18 (MA1819 diluted 1:100; ThermoFisher Scientific, Waltham, PA, USA), and human nucleoli (ab190710 diluted 1:100; Abcam).

Techniques: Labeling

Journal: Investigative Ophthalmology & Visual Science

Article Title: Myo/Nog Cells Give Rise to Myofibroblasts During Epiretinal Membrane Formation in a Mouse Model of Proliferative Vitreoretinopathy

doi: 10.1167/iovs.64.2.1

Figure Lengend Snippet: Localization of BAI1 and Noggin in ERMs and retina. Sections of mouse eyes with PVR grades 0 ( A-C ), 5 ( D-F ), and 3 ( G-I ) were double labeled with antibodies to BAI1 ( red ) and noggin ( green ). Nuclei were stained with DAPI ( blue ). The overlap of red and green appears yellow in merged images. BAI1 and noggin were co-localized in a small number of cells in the grade 0 retina A to - C . BAI1+/noggin+ cells were present throughout the ERM D to F and throughout the retina G to I . GCL = ganglion cell layer; IPL = inner plexiform layer; INL = inner nuclear layer; OPL = outer plexiform layer; ONL = outer nuclear layer. Bar = 9 µM.

Article Snippet: Myo/Nog cells were identified in tissue sections by double labeling with the anti-BAI1 G8 IgM mouse mAb and the anti-noggin goat polyclonal anti-serum (AF719; R&D Systems, Minneapolis, MN, USA), as described previously., Double labeling also was carried out with the IgM BAI1 mAb and IgG mAb antibodies to α-SMA (F3777 diluted 1:250; Sigma-Aldrich, St. Louis, MO, USA), striated muscle myosin II (ab58899 diluted 1:200; Abcam, Cambridge, MA, USA), the leukocyte markers CD68 (ab201340 diluted 1:200; Abcam), CD45 (ab10558 diluted 1:200; Abcam), and CD18 (MA1819 diluted 1:100; ThermoFisher Scientific, Waltham, PA, USA), and human nucleoli (ab190710 diluted 1:100; Abcam).

Techniques: Labeling, Staining

Journal: Investigative Ophthalmology & Visual Science

Article Title: Myo/Nog Cells Give Rise to Myofibroblasts During Epiretinal Membrane Formation in a Mouse Model of Proliferative Vitreoretinopathy

doi: 10.1167/iovs.64.2.1

Figure Lengend Snippet: Number of BAI1+ cells per grade in a mouse model of PVR. Tissue sections of the eye were fluorescently labeled with the anti-BAI1 G8 mAb. The numbers of BAI1+ cells were counted in each section on the inner retinal surface (IRS; (grade 2), epiretinal membrane (ERM; grades 3–6), and retina (grades 0–6) ( A ), and the cornea, ciliary body, lens, and zonules of Zinn (grades 0–6) ( B ). The values are the mean ± standard deviation (SD) of the number of BAI1+ cells/section. The numbers of sections labeled with the BAI1 mAb are indicated above the SD bar. Grade 0 (injected with PBS): 9 eyes; grades 1/2: 7 eyes; grades 3/4 10 eyes; and grades 5/6: 13 eyes. * Indicates P values ≤ 0.004.

Article Snippet: Myo/Nog cells were identified in tissue sections by double labeling with the anti-BAI1 G8 IgM mouse mAb and the anti-noggin goat polyclonal anti-serum (AF719; R&D Systems, Minneapolis, MN, USA), as described previously., Double labeling also was carried out with the IgM BAI1 mAb and IgG mAb antibodies to α-SMA (F3777 diluted 1:250; Sigma-Aldrich, St. Louis, MO, USA), striated muscle myosin II (ab58899 diluted 1:200; Abcam, Cambridge, MA, USA), the leukocyte markers CD68 (ab201340 diluted 1:200; Abcam), CD45 (ab10558 diluted 1:200; Abcam), and CD18 (MA1819 diluted 1:100; ThermoFisher Scientific, Waltham, PA, USA), and human nucleoli (ab190710 diluted 1:100; Abcam).

Techniques: Labeling, Membrane, Standard Deviation, Injection

Journal: Investigative Ophthalmology & Visual Science

Article Title: Myo/Nog Cells Give Rise to Myofibroblasts During Epiretinal Membrane Formation in a Mouse Model of Proliferative Vitreoretinopathy

doi: 10.1167/iovs.64.2.1

Figure Lengend Snippet: Localization of BAI1 and muscle proteins in ERMs and the retina. Sections of mouse eyes with PVR grades 5/6 were double labeled with antibodies to BAI1 ( red ) and α-SMA ( green ) ( A-H ) or striated muscle myosin ( green ) ( I-P ). Nuclei were stained with DAPI ( blue ). The overlap of red and green appears yellow in merged images ( D, H, L , P ). Regions within the boxes of the low magnification images in A , E , I , and M are shown at higher magnification in B to D , F to H , J to L , and N to P , respectively. BAI1 and α-SMA were co-localized in the ERM located in the vicinity of a retina fold ( arrow in A ). BAI1+/ α-SMA+ cells also were present throughout the layers of the retina ( E-H ). BAI1+/myosin+ cells were located within the ERM above the retinal folds ( white arrows in I , J - L ) and in the retina ( M - P ). ONL = outer nuclear layer; OPL = outer plexiform layer; INL = inner nuclear layer; IPL = inner plexiform layer; GCL = ganglion cell layer. Bar = 9 µM in A to D , F to H , J to L , and N and O , 57 µM in I and M , and 135 µM in E .

Article Snippet: Myo/Nog cells were identified in tissue sections by double labeling with the anti-BAI1 G8 IgM mouse mAb and the anti-noggin goat polyclonal anti-serum (AF719; R&D Systems, Minneapolis, MN, USA), as described previously., Double labeling also was carried out with the IgM BAI1 mAb and IgG mAb antibodies to α-SMA (F3777 diluted 1:250; Sigma-Aldrich, St. Louis, MO, USA), striated muscle myosin II (ab58899 diluted 1:200; Abcam, Cambridge, MA, USA), the leukocyte markers CD68 (ab201340 diluted 1:200; Abcam), CD45 (ab10558 diluted 1:200; Abcam), and CD18 (MA1819 diluted 1:100; ThermoFisher Scientific, Waltham, PA, USA), and human nucleoli (ab190710 diluted 1:100; Abcam).

Techniques: Labeling, Staining

Journal: Investigative Ophthalmology & Visual Science

Article Title: Myo/Nog Cells Give Rise to Myofibroblasts During Epiretinal Membrane Formation in a Mouse Model of Proliferative Vitreoretinopathy

doi: 10.1167/iovs.64.2.1

Figure Lengend Snippet: Localization of Myo/Nog, human, and pigmented cells in PVR . Sections of mouse eyes with PVR were double labeled with antibodies to BAI1 and hNuc ( A, C-E ), hNuc, and TUNEL reagents ( B ), and BAI1 and striated muscle myosin ( F-H , R-U ), noggin ( J-M ), or α-SMA ( N-Q ). The colors of the fluorescent secondary antibodies are indicated in the annotations. Nuclei were stained with DAPI ( blue ). The overlap of red and green appears yellow in merged images ( B , L , M , P , Q , R-U ). G to - I , M , Q , S , and U are merges of red , green , and/or blue and DIC. HNuc+ cells in the vitreous of grades 1/2 PVR were not labeled with the BAI1 mAb A . Some hNuc+ cells were apoptotic B . BAI1+ cells lacked detectable levels of hNuc in the ERM C . One hNuc+ nucleus appeared to be present in a cell with two other unlabeled nuclei ( arrow in C , enlarged in D and E ). BAI1 staining was between the three nuclei ( D , E ). BAI1 and pigment were present in a cell with a normal nucleus and a dysmorphic nucleus ( F , G ). A pigmented cell on the surface of a grade 5/6 ERM was surrounded by BAI1+/myosin- cells ( arrow in H ). A low magnification image shows pigmented cells that had invaded the retina ( arrow in I ). BAI1+/noggin+ cells in the grades 5/6 ERM did not contain pigment ( K-M ). Pigmented cells were visible on the zonule ( arrow in M ). BAI1 and α-SMA were co-localized in the ERM ( red arrow in P ) and on the internal and external surface of the lens capsule ( N-Q , white arrows in P ). Cells with pigment were present on the surface of the ERM ( arrows in Q ). Pigmented cells surrounded an aggregate of BAI1+/myosin+ cells in the retina ( R , S ) and were interspersed with differentiated Myo/Nog cells in grades 5/6 ERMs ( T , U ). INL = inner nuclear layer; IPL = inner plexiform layer; GCL = ganglion cell layer; Z = zonule. Bar = 9 µM in A-C, F-I and K-N, 10 µM in G and H; 57 µM in J, and 12 µM in O-R.

Article Snippet: Myo/Nog cells were identified in tissue sections by double labeling with the anti-BAI1 G8 IgM mouse mAb and the anti-noggin goat polyclonal anti-serum (AF719; R&D Systems, Minneapolis, MN, USA), as described previously., Double labeling also was carried out with the IgM BAI1 mAb and IgG mAb antibodies to α-SMA (F3777 diluted 1:250; Sigma-Aldrich, St. Louis, MO, USA), striated muscle myosin II (ab58899 diluted 1:200; Abcam, Cambridge, MA, USA), the leukocyte markers CD68 (ab201340 diluted 1:200; Abcam), CD45 (ab10558 diluted 1:200; Abcam), and CD18 (MA1819 diluted 1:100; ThermoFisher Scientific, Waltham, PA, USA), and human nucleoli (ab190710 diluted 1:100; Abcam).

Techniques: Labeling, TUNEL Assay, Staining

Journal: Investigative Ophthalmology & Visual Science

Article Title: Myo/Nog Cells Give Rise to Myofibroblasts During Epiretinal Membrane Formation in a Mouse Model of Proliferative Vitreoretinopathy

doi: 10.1167/iovs.64.2.1

Figure Lengend Snippet: Number of Cells Labeled With Antibodies to Leukocyte Markers in PVR

Article Snippet: Myo/Nog cells were identified in tissue sections by double labeling with the anti-BAI1 G8 IgM mouse mAb and the anti-noggin goat polyclonal anti-serum (AF719; R&D Systems, Minneapolis, MN, USA), as described previously., Double labeling also was carried out with the IgM BAI1 mAb and IgG mAb antibodies to α-SMA (F3777 diluted 1:250; Sigma-Aldrich, St. Louis, MO, USA), striated muscle myosin II (ab58899 diluted 1:200; Abcam, Cambridge, MA, USA), the leukocyte markers CD68 (ab201340 diluted 1:200; Abcam), CD45 (ab10558 diluted 1:200; Abcam), and CD18 (MA1819 diluted 1:100; ThermoFisher Scientific, Waltham, PA, USA), and human nucleoli (ab190710 diluted 1:100; Abcam).

Techniques: Labeling

Journal: Frontiers in Neuroscience

Article Title: Acute Response and Neuroprotective Role of Myo/Nog Cells Assessed in a Rat Model of Focal Brain Injury

doi: 10.3389/fnins.2021.780707

Figure Lengend Snippet: Identification of the NS injury tract lesion site, Myo/Nog cells and dying neurons in the rat brain. The area where the needle is inserted into parietal cortex and hippocampus is shown in the hematoxylin and eosin stained section in (A) (dashed line). The NS tract and the counting area within 1 mm of the lesion is shown in (B) . Sections were double labeled with antibodies to BAI1 (green) and TUNEL reagents (red), BAI1 (green), and Noggin (red) (D,E) , and NeuN (green) (F) and TUNEL (red) (C) . Nuclei were stained with DAPI in (B–F) . Overlap of green and red appear yellow in merged images. Unmerged images are shown as insets at the bottom of the photographs in (C–E) . Images were acquired from the uninjured posterior parietal cortex and hippocampus (A) , along the NS tract within the parietal cortex (B,D) , interface of the parietal cortex and hippocampus (C,F) , and the end of the NS tract in the hippocampus (E) . A BAI1+ Myo/Nog cell appears to have phagocytosed a TUNEL+ cell (C) . The red arrows in (C) depict separate nuclei. Myo/Nog cells co-expressed BAI1 and Noggin (D,E) . The majority of TUNEL+ cells were NeuN+ neurons (F) .

Article Snippet: Sections were double labeled with the BAI1 mAb and a goat anti-mouse polyclonal antiserum to Noggin diluted 1:200 (R&D Systems, Minneapolis, MN, United States).

Techniques: Staining, Labeling, TUNEL Assay

Journal: Frontiers in Neuroscience

Article Title: Acute Response and Neuroprotective Role of Myo/Nog Cells Assessed in a Rat Model of Focal Brain Injury

doi: 10.3389/fnins.2021.780707

Figure Lengend Snippet: Comparison of the number of Myo/Nog cells before and after NS injury and with depletion or addition of BAI1+ cells. Six tissue sections from 3 to 6 animals per treatment group were labeled with the BAI1 mAb (green in B–D ). Nuclei were stained with DAPI (blue in B–D ). BAI1+ cells were counted within a 1 mm area lateral to NS tract or in a similar area in the uninjured brain. The results are the means ± SEM 24 h after NS with injection of PBS, the BAI1 mAb and complement (BAI1ab/comp), complement alone (comp), BAI1+ cells or BAI1– cells (A) . NS injury significantly increased the number of Myo/Nog cells compared to a similar region in the uninjured brain ( ** p = 0.00001). Treatment with the BAI1 mAb and complement (abBAI1/comp) significantly reduced the number of Myo/Nog cells compared to NS with PBS ( p = 0.002), BAI1+ cells ( p = 0.00001), BAI1– cells ( p = 0.0002), or comp alone ( p = 0.04). Significantly more Myo/Nog cells were present in brains injected with Myo/Nog cells than the other groups (* p < 0.05). Myo/Nog cells were most prevalent after injection of exogenous BAI1+ cells ( D ; p = 0.00006). Unmerged images are shown as insets at the bottom of the photographs of merged images. Panels (B–D) are photographs of BAI1 and DAPI labeling in the parietal cortex of uninjured brain and NS injured brains injected with PBS or BAI1+ cells. More Myo/Nog cells were observed after injecting BAI1+ cells than in uninjured brains and those injected with PBS.

Article Snippet: Sections were double labeled with the BAI1 mAb and a goat anti-mouse polyclonal antiserum to Noggin diluted 1:200 (R&D Systems, Minneapolis, MN, United States).

Techniques: Comparison, Labeling, Staining, Injection

Journal: Frontiers in Neuroscience

Article Title: Acute Response and Neuroprotective Role of Myo/Nog Cells Assessed in a Rat Model of Focal Brain Injury

doi: 10.3389/fnins.2021.780707

Figure Lengend Snippet: Comparison of cell death before and after NS injury and with depletion or addition of BAI1+ cells. Six tissue sections from 3 to 6 animals per treatment group were stained with TUNEL reagents (red in B–D ). Nuclei were stained with DAPI (blue in B–D ). The number of TUNEL+ cells was counted within a 1 mm area lateral to NS tract or in a similar area in the uninjured brain. The results are the means ± SEM 24 h after NS with injection of PBS, the BAI1 mAb and complement (BAI1ab/comp), complement alone (comp), BAI1+ cells or BAI1– cells (A) . Significantly fewer TUNEL+ cells were present in uninjured tissue than the other treatment groups (* p = 0.00001). NS injury significantly increased the number of TUNEL+ cells in all treatment groups ( p < 0.05). Injection of BAI1+ cells isolated from the brains of other animals significantly reduced the number of TUNEL+ cells compared to all other groups ( ** p < 0.05). The numbers of TUNEL+ cells were similar in brains injected with PBS, BAI1 and complement (BAI1Ab/comp), and complement alone (Comp). Panels (B–D) are photographs of TUNEL and DAPI labeling in the parietal cortex of the uninjured brain and NS injured brains injected with PBS or BAI1+ cells. Unmerged images are shown as insets at the bottom of the photographs of merged images. TUNEL+ cells were rare in the uninjured brain (B) . Fewer TUNEL+ cells were observed after injecting BAI1+ cells (D) than in those injected with PBS (C) .

Article Snippet: Sections were double labeled with the BAI1 mAb and a goat anti-mouse polyclonal antiserum to Noggin diluted 1:200 (R&D Systems, Minneapolis, MN, United States).

Techniques: Comparison, Staining, TUNEL Assay, Injection, Isolation, Labeling

Journal: Frontiers in Neuroscience

Article Title: Acute Response and Neuroprotective Role of Myo/Nog Cells Assessed in a Rat Model of Focal Brain Injury

doi: 10.3389/fnins.2021.780707

Figure Lengend Snippet: Comparison of the number of NeuN+ neurons before and after NS injury and with depletion or addition of BAI1+ cells. Six tissue sections from 3 to 6 animals per treatment group were stained with TUNEL reagents (red in B–D ). Nuclei were stained with DAPI (blue in B–D ). NeuN positive cells were counted within a 1 mm area lateral to NS tract or in a similar area in the uninjured brain. The results are the means ± SEM 24 h after NS with injection of PBS, the BAI1 mAb and complement (BAI1ab/comp), complement alone (comp), BAI1+ cells or BAI1– cells (A) . The elevation in the number of NeuN cells in brains injected with BAI1+ compared to the uninjured brain was not statistically significant ( p = 0.06). The number of NeuN+ cells was significantly greater after injection of BAI1+ cells than in the other treatment groups (* p ≤ 0.05). Panels (B–D) are photographs of NeuN and DAPI Unmerged images are shown as insets at the bottom of the photographs of merged images.

Article Snippet: Sections were double labeled with the BAI1 mAb and a goat anti-mouse polyclonal antiserum to Noggin diluted 1:200 (R&D Systems, Minneapolis, MN, United States).

Techniques: Comparison, Staining, TUNEL Assay, Injection

Journal: Frontiers in Neuroscience

Article Title: Acute Response and Neuroprotective Role of Myo/Nog Cells Assessed in a Rat Model of Focal Brain Injury

doi: 10.3389/fnins.2021.780707

Figure Lengend Snippet: Identification of the NS injury tract lesion site, Myo/Nog cells and dying neurons in the rat brain. The area where the needle is inserted into parietal cortex and hippocampus is shown in the hematoxylin and eosin stained section in (A) (dashed line). The NS tract and the counting area within 1 mm of the lesion is shown in (B) . Sections were double labeled with antibodies to BAI1 (green) and TUNEL reagents (red), BAI1 (green), and Noggin (red) (D,E) , and NeuN (green) (F) and TUNEL (red) (C) . Nuclei were stained with DAPI in (B–F) . Overlap of green and red appear yellow in merged images. Unmerged images are shown as insets at the bottom of the photographs in (C–E) . Images were acquired from the uninjured posterior parietal cortex and hippocampus (A) , along the NS tract within the parietal cortex (B,D) , interface of the parietal cortex and hippocampus (C,F) , and the end of the NS tract in the hippocampus (E) . A BAI1+ Myo/Nog cell appears to have phagocytosed a TUNEL+ cell (C) . The red arrows in (C) depict separate nuclei. Myo/Nog cells co-expressed BAI1 and Noggin (D,E) . The majority of TUNEL+ cells were NeuN+ neurons (F) .

Article Snippet: Remaining cells were incubated with the anti-BAI1 mAb and magnetic anti-IgM secondary antibodies (Miltenyi Biotec).

Techniques: Staining, Labeling, TUNEL Assay

Journal: Frontiers in Neuroscience

Article Title: Acute Response and Neuroprotective Role of Myo/Nog Cells Assessed in a Rat Model of Focal Brain Injury

doi: 10.3389/fnins.2021.780707

Figure Lengend Snippet: Comparison of the number of Myo/Nog cells before and after NS injury and with depletion or addition of BAI1+ cells. Six tissue sections from 3 to 6 animals per treatment group were labeled with the BAI1 mAb (green in B–D ). Nuclei were stained with DAPI (blue in B–D ). BAI1+ cells were counted within a 1 mm area lateral to NS tract or in a similar area in the uninjured brain. The results are the means ± SEM 24 h after NS with injection of PBS, the BAI1 mAb and complement (BAI1ab/comp), complement alone (comp), BAI1+ cells or BAI1– cells (A) . NS injury significantly increased the number of Myo/Nog cells compared to a similar region in the uninjured brain ( ** p = 0.00001). Treatment with the BAI1 mAb and complement (abBAI1/comp) significantly reduced the number of Myo/Nog cells compared to NS with PBS ( p = 0.002), BAI1+ cells ( p = 0.00001), BAI1– cells ( p = 0.0002), or comp alone ( p = 0.04). Significantly more Myo/Nog cells were present in brains injected with Myo/Nog cells than the other groups (* p < 0.05). Myo/Nog cells were most prevalent after injection of exogenous BAI1+ cells ( D ; p = 0.00006). Unmerged images are shown as insets at the bottom of the photographs of merged images. Panels (B–D) are photographs of BAI1 and DAPI labeling in the parietal cortex of uninjured brain and NS injured brains injected with PBS or BAI1+ cells. More Myo/Nog cells were observed after injecting BAI1+ cells than in uninjured brains and those injected with PBS.

Article Snippet: Remaining cells were incubated with the anti-BAI1 mAb and magnetic anti-IgM secondary antibodies (Miltenyi Biotec).

Techniques: Comparison, Labeling, Staining, Injection

Journal: Frontiers in Neuroscience

Article Title: Acute Response and Neuroprotective Role of Myo/Nog Cells Assessed in a Rat Model of Focal Brain Injury

doi: 10.3389/fnins.2021.780707

Figure Lengend Snippet: Comparison of cell death before and after NS injury and with depletion or addition of BAI1+ cells. Six tissue sections from 3 to 6 animals per treatment group were stained with TUNEL reagents (red in B–D ). Nuclei were stained with DAPI (blue in B–D ). The number of TUNEL+ cells was counted within a 1 mm area lateral to NS tract or in a similar area in the uninjured brain. The results are the means ± SEM 24 h after NS with injection of PBS, the BAI1 mAb and complement (BAI1ab/comp), complement alone (comp), BAI1+ cells or BAI1– cells (A) . Significantly fewer TUNEL+ cells were present in uninjured tissue than the other treatment groups (* p = 0.00001). NS injury significantly increased the number of TUNEL+ cells in all treatment groups ( p < 0.05). Injection of BAI1+ cells isolated from the brains of other animals significantly reduced the number of TUNEL+ cells compared to all other groups ( ** p < 0.05). The numbers of TUNEL+ cells were similar in brains injected with PBS, BAI1 and complement (BAI1Ab/comp), and complement alone (Comp). Panels (B–D) are photographs of TUNEL and DAPI labeling in the parietal cortex of the uninjured brain and NS injured brains injected with PBS or BAI1+ cells. Unmerged images are shown as insets at the bottom of the photographs of merged images. TUNEL+ cells were rare in the uninjured brain (B) . Fewer TUNEL+ cells were observed after injecting BAI1+ cells (D) than in those injected with PBS (C) .

Article Snippet: Remaining cells were incubated with the anti-BAI1 mAb and magnetic anti-IgM secondary antibodies (Miltenyi Biotec).

Techniques: Comparison, Staining, TUNEL Assay, Injection, Isolation, Labeling

Journal: Frontiers in Neuroscience

Article Title: Acute Response and Neuroprotective Role of Myo/Nog Cells Assessed in a Rat Model of Focal Brain Injury

doi: 10.3389/fnins.2021.780707

Figure Lengend Snippet: Comparison of the number of NeuN+ neurons before and after NS injury and with depletion or addition of BAI1+ cells. Six tissue sections from 3 to 6 animals per treatment group were stained with TUNEL reagents (red in B–D ). Nuclei were stained with DAPI (blue in B–D ). NeuN positive cells were counted within a 1 mm area lateral to NS tract or in a similar area in the uninjured brain. The results are the means ± SEM 24 h after NS with injection of PBS, the BAI1 mAb and complement (BAI1ab/comp), complement alone (comp), BAI1+ cells or BAI1– cells (A) . The elevation in the number of NeuN cells in brains injected with BAI1+ compared to the uninjured brain was not statistically significant ( p = 0.06). The number of NeuN+ cells was significantly greater after injection of BAI1+ cells than in the other treatment groups (* p ≤ 0.05). Panels (B–D) are photographs of NeuN and DAPI Unmerged images are shown as insets at the bottom of the photographs of merged images.

Article Snippet: Remaining cells were incubated with the anti-BAI1 mAb and magnetic anti-IgM secondary antibodies (Miltenyi Biotec).

Techniques: Comparison, Staining, TUNEL Assay, Injection

Journal: PLoS ONE

Article Title: Brain-specific angiogenesis inhibitor 1 is expressed in the Myo/Nog cell lineage

doi: 10.1371/journal.pone.0234792

Figure Lengend Snippet: Tissue sources and numbers of tissue and tissue sections screened by immunofluorescence localization of antibodies.

Article Snippet: Double labeling also was performed with the R&D BAI1 mAb and the anti-Noggin goat polyclonal antiserum (AF719; R&D Systems), anti-MyoD IgG1 mAb (MA5-12902, ThermoFisher Scientific, Rockford, IL), anti-MyoD rabbit polyclonal antiserum (ab203383, Abcam, Cambridge, MA), anti-NeuN IgG mAb (ab104224, Abcam), anti-GFAP IgG mAb (ab10062, Abcam) and anti-Iba1 rabbit mAb (ab178846, Abcam), all diluted 1:100.

Techniques: Immunofluorescence

Journal: PLoS ONE

Article Title: Brain-specific angiogenesis inhibitor 1 is expressed in the Myo/Nog cell lineage

doi: 10.1371/journal.pone.0234792

Figure Lengend Snippet: The G8 mAb was used to screen clones of HEK-239T cells expressing a single human membrane protein. G8 binding was detected by flow cytometry. A. Binding values were normalized and transformed to give a single numerical value for binding of the G8 mAb against each target protein (normalized target binding). Non-specific fluorescence was determined to be any value below three standard deviations above noise. G8 binding above background occurred with the clone expressing BAI1. B. Validation of binding of the G8 mAb to BAI1 or vector alone was carried out with HEK-293T cells transfected with the plasmid construct expressing target using a serial dilution of mAb. C. Binding of different concentrations of the G8 mAb was calculated as the signal to background ratio of fluorescence from the BAI1 expressing clone to empty vector clone.

Article Snippet: Double labeling also was performed with the R&D BAI1 mAb and the anti-Noggin goat polyclonal antiserum (AF719; R&D Systems), anti-MyoD IgG1 mAb (MA5-12902, ThermoFisher Scientific, Rockford, IL), anti-MyoD rabbit polyclonal antiserum (ab203383, Abcam, Cambridge, MA), anti-NeuN IgG mAb (ab104224, Abcam), anti-GFAP IgG mAb (ab10062, Abcam) and anti-Iba1 rabbit mAb (ab178846, Abcam), all diluted 1:100.

Techniques: Clone Assay, Expressing, Membrane, Binding Assay, Flow Cytometry, Transformation Assay, Fluorescence, Plasmid Preparation, Transfection, Construct, Serial Dilution

Journal: PLoS ONE

Article Title: Brain-specific angiogenesis inhibitor 1 is expressed in the Myo/Nog cell lineage

doi: 10.1371/journal.pone.0234792

Figure Lengend Snippet: Plates were coated with recombinant human BAI1 protein produced by CHO cells. Binding of the G8 IgM mAb and R&D BAI1 IgG mAb (a-BAI) was detected with anti-mouse IgM and IgG affinity purified F(ab’)2 secondary antibodies, respectively, conjugated with horseradish peroxidase. Both mAbs bound to BAI1 protein.

Article Snippet: Double labeling also was performed with the R&D BAI1 mAb and the anti-Noggin goat polyclonal antiserum (AF719; R&D Systems), anti-MyoD IgG1 mAb (MA5-12902, ThermoFisher Scientific, Rockford, IL), anti-MyoD rabbit polyclonal antiserum (ab203383, Abcam, Cambridge, MA), anti-NeuN IgG mAb (ab104224, Abcam), anti-GFAP IgG mAb (ab10062, Abcam) and anti-Iba1 rabbit mAb (ab178846, Abcam), all diluted 1:100.

Techniques: Recombinant, Produced, Binding Assay, Affinity Purification

Journal: PLoS ONE

Article Title: Brain-specific angiogenesis inhibitor 1 is expressed in the Myo/Nog cell lineage

doi: 10.1371/journal.pone.0234792

Figure Lengend Snippet: An alanine-scan library of BAI1 was constructed. The G8 and reference 5D6 mAbs were screened for binding to each individual BAI1 variant. A. Identification of candidate critical clones for G8 mAb binding was determined in duplicate by high throughput flow cytometry. For each point, background fluorescence was subtracted from the raw data that were then normalized to G8 reactivity with wild type BAI1. For each mutant clone, the mean binding value was plotted as a function of expression represented by control antibody reactivity. Candidate critical clones (red circles) were identified with a threshold (dashed line) of <40% binding of the G8 mAb and >50% of binding of the control 5D6 mAb to wild type BAI1. B. Identification of validated critical residues for G8 mAb binding. Candidate critical residues for G8 were rescreened in quadruplicate and the mean binding reactivities obtained. Validated critical residues for G8 binding (outlined in red) were residues whose mutations that resulted in <20% of wild type binding but positive (>70% binding) for binding of the 5D6 control mAb. Additional validated secondary residues (outlined in blue) were identified that did not meet the threshold guidelines but whose decreased binding activity and proximity to critical residues suggested that they may be part of the antibody epitope. C. Visualization of validated critical residues for G8 mAb binding. Critical residues (red spheres) and secondary residues (blue spheres) which may also contribute to binding were visualized on two Phyre-generated model structures that were combined to model BAI1 residues 262–934. The G8 critical residues were visualized on a model of the BAI1 thrombospondin repeat domains based on the structure of properdin (PDB ID# 1WOR; .

Article Snippet: Double labeling also was performed with the R&D BAI1 mAb and the anti-Noggin goat polyclonal antiserum (AF719; R&D Systems), anti-MyoD IgG1 mAb (MA5-12902, ThermoFisher Scientific, Rockford, IL), anti-MyoD rabbit polyclonal antiserum (ab203383, Abcam, Cambridge, MA), anti-NeuN IgG mAb (ab104224, Abcam), anti-GFAP IgG mAb (ab10062, Abcam) and anti-Iba1 rabbit mAb (ab178846, Abcam), all diluted 1:100.

Techniques: Construct, Binding Assay, Variant Assay, Clone Assay, High Throughput Screening Assay, Flow Cytometry, Fluorescence, Mutagenesis, Expressing, Control, Activity Assay, Generated

Journal: PLoS ONE

Article Title: Brain-specific angiogenesis inhibitor 1 is expressed in the Myo/Nog cell lineage

doi: 10.1371/journal.pone.0234792

Figure Lengend Snippet: Localization of antibodies to BAI1, Noggin and MyoD in the skin, eyes and brain. Human lens tissue and tissue sections of human tattooed skin, rabbit anterior cavity, rat retina and mouse brain were double labeled with the G8 and R&D BAI1 mAbs and antibodies to Noggin and MyoD. The total numbers of double labeled cells were counted in a subset of tissue sections. The total numbers of single labeled cells were quantified in all sections with the exception of MyoD-/R&D BAI1 mAb+ and MyoD-/G8- cells that were counted in a subset of skin and anterior cavity sections. The numbers of tissues and sections scored are indicated in parenthesis. The results are the mean ± standard deviation. The R&D BAI1 mAb co-localized with G8 and Noggin with rare exception. BAI1+ cells without MyoD were present in the skin and anterior cavity.

Article Snippet: Double labeling also was performed with the R&D BAI1 mAb and the anti-Noggin goat polyclonal antiserum (AF719; R&D Systems), anti-MyoD IgG1 mAb (MA5-12902, ThermoFisher Scientific, Rockford, IL), anti-MyoD rabbit polyclonal antiserum (ab203383, Abcam, Cambridge, MA), anti-NeuN IgG mAb (ab104224, Abcam), anti-GFAP IgG mAb (ab10062, Abcam) and anti-Iba1 rabbit mAb (ab178846, Abcam), all diluted 1:100.

Techniques: Labeling, Standard Deviation

Journal: PLoS ONE

Article Title: Brain-specific angiogenesis inhibitor 1 is expressed in the Myo/Nog cell lineage

doi: 10.1371/journal.pone.0234792

Figure Lengend Snippet: Tissue sections of human tattooed skin (A-C), human anterior lens tissue (D-F), rabbit eyes (G-I) and rat retina (J-L) were double labeled with the G8 (red) and BAI1 (green) mAbs. Nuclei were stained with Hoechst dye (blue). Unmerged images are shown in A, B, D, E and the insets in G-I and K and L. Overlap of red and green appear yellow in triple merged images in C, F, G-I, K and L. A merge of fluorescence and DIC is shown in the inset of C. The G8 and BAI1 mAbs labeled the same cells in the skin, human lens, rabbit lens (G), ciliary body (H) and cornea (I), and the inner plexiform and inner nuclear layers of the mouse retina (K and L). Minimal background fluorescence was observed in the anti-IgM and anti-IgG secondary antibody controls for the skin (M), human lens tissue (N), rabbit lens (O) and mouse retina P and Q. Bar = 9 μM in A-I and K-Q and 54 μM in J.

Article Snippet: Double labeling also was performed with the R&D BAI1 mAb and the anti-Noggin goat polyclonal antiserum (AF719; R&D Systems), anti-MyoD IgG1 mAb (MA5-12902, ThermoFisher Scientific, Rockford, IL), anti-MyoD rabbit polyclonal antiserum (ab203383, Abcam, Cambridge, MA), anti-NeuN IgG mAb (ab104224, Abcam), anti-GFAP IgG mAb (ab10062, Abcam) and anti-Iba1 rabbit mAb (ab178846, Abcam), all diluted 1:100.

Techniques: Labeling, Staining, Fluorescence

Journal: PLoS ONE

Article Title: Brain-specific angiogenesis inhibitor 1 is expressed in the Myo/Nog cell lineage

doi: 10.1371/journal.pone.0234792

Figure Lengend Snippet: Tissue sections of human tattooed skin (A and B), human anterior lens tissue (C) and rabbit anterior cavity (D-I) were double labeled with the R&D BAI1 mAb and antibodies to noggin or MyoD. The colors of the fluorescent secondary antibodies are indicated in each photograph. Overlap of red and green appears yellow in merged images. Nuclei were stained with Hoechst dye. Double labeled cells were present in the skin (A and B), anterior human lens tissue (C) and the rabbit lens (D and G), ciliary body (E and H) and cornea (F and I). Bar = 9 μM.

Article Snippet: Double labeling also was performed with the R&D BAI1 mAb and the anti-Noggin goat polyclonal antiserum (AF719; R&D Systems), anti-MyoD IgG1 mAb (MA5-12902, ThermoFisher Scientific, Rockford, IL), anti-MyoD rabbit polyclonal antiserum (ab203383, Abcam, Cambridge, MA), anti-NeuN IgG mAb (ab104224, Abcam), anti-GFAP IgG mAb (ab10062, Abcam) and anti-Iba1 rabbit mAb (ab178846, Abcam), all diluted 1:100.

Techniques: Labeling, Staining

Journal: PLoS ONE

Article Title: Brain-specific angiogenesis inhibitor 1 is expressed in the Myo/Nog cell lineage

doi: 10.1371/journal.pone.0234792

Figure Lengend Snippet: Tissue sections from the day-10 mouse brain were stained with hematoxylin and eosin (sagittal sections A and D; coronal sections B and C) or double labeled with the G8 and the R&D BAI1 mAbs, or G8 and antibodies to Iba1, NeuN or GFAP. The areas within the boxes of the H&E stained sections are shown at high magnification in the fluorescence photomicrographs. The colors of the fluorescent secondary antibodies are indicated in the unmerged photographs (E, F, H and I). Nuclei were stained with Hoechst dye. Overlap of red and green, when present, appears yellow in merged images (G, J, K, L and M). The G8 and BAI1 mAbs labeled the same subpopulation of cells in the hippocampal formation (box in A; E-G). The Noggin and BAI1 antibodies also bound to the same cells in the glomerular layer of the olfactory bulb (box in B; H-J). G8 did not co-localize with Iba1 (K, from box in A), NeuN (L from lower box in C) or GFAP (M from box in D). Minimal fluorescence was observed with the anti-IgM and anti-IgG (inset in G) or the anti-goat and anti-IgG (inset in J) secondary antibodies. Bar = 270 μM in A-D and 9 μM in E-M.

Article Snippet: Double labeling also was performed with the R&D BAI1 mAb and the anti-Noggin goat polyclonal antiserum (AF719; R&D Systems), anti-MyoD IgG1 mAb (MA5-12902, ThermoFisher Scientific, Rockford, IL), anti-MyoD rabbit polyclonal antiserum (ab203383, Abcam, Cambridge, MA), anti-NeuN IgG mAb (ab104224, Abcam), anti-GFAP IgG mAb (ab10062, Abcam) and anti-Iba1 rabbit mAb (ab178846, Abcam), all diluted 1:100.

Techniques: Staining, Labeling, Fluorescence

Journal: PLoS ONE

Article Title: Brain-specific angiogenesis inhibitor 1 is expressed in the Myo/Nog cell lineage

doi: 10.1371/journal.pone.0234792

Figure Lengend Snippet: Tissue sources and numbers of tissue and tissue sections screened by immunofluorescence localization of antibodies.

Article Snippet: Both G8 and anti-BAI1 mAb from R&D Systems (R&D BAI1) bound to a substrate of purified BAI1 protein containing amino acids 31–879 located within the extracellular domain ( ).

Techniques: Immunofluorescence

Journal: PLoS ONE

Article Title: Brain-specific angiogenesis inhibitor 1 is expressed in the Myo/Nog cell lineage

doi: 10.1371/journal.pone.0234792

Figure Lengend Snippet: The G8 mAb was used to screen clones of HEK-239T cells expressing a single human membrane protein. G8 binding was detected by flow cytometry. A. Binding values were normalized and transformed to give a single numerical value for binding of the G8 mAb against each target protein (normalized target binding). Non-specific fluorescence was determined to be any value below three standard deviations above noise. G8 binding above background occurred with the clone expressing BAI1. B. Validation of binding of the G8 mAb to BAI1 or vector alone was carried out with HEK-293T cells transfected with the plasmid construct expressing target using a serial dilution of mAb. C. Binding of different concentrations of the G8 mAb was calculated as the signal to background ratio of fluorescence from the BAI1 expressing clone to empty vector clone.

Article Snippet: Both G8 and anti-BAI1 mAb from R&D Systems (R&D BAI1) bound to a substrate of purified BAI1 protein containing amino acids 31–879 located within the extracellular domain ( ).

Techniques: Clone Assay, Expressing, Membrane, Binding Assay, Flow Cytometry, Transformation Assay, Fluorescence, Plasmid Preparation, Transfection, Construct, Serial Dilution

Journal: PLoS ONE

Article Title: Brain-specific angiogenesis inhibitor 1 is expressed in the Myo/Nog cell lineage

doi: 10.1371/journal.pone.0234792

Figure Lengend Snippet: Plates were coated with recombinant human BAI1 protein produced by CHO cells. Binding of the G8 IgM mAb and R&D BAI1 IgG mAb (a-BAI) was detected with anti-mouse IgM and IgG affinity purified F(ab’)2 secondary antibodies, respectively, conjugated with horseradish peroxidase. Both mAbs bound to BAI1 protein.

Article Snippet: Both G8 and anti-BAI1 mAb from R&D Systems (R&D BAI1) bound to a substrate of purified BAI1 protein containing amino acids 31–879 located within the extracellular domain ( ).

Techniques: Recombinant, Produced, Binding Assay, Affinity Purification

Journal: PLoS ONE

Article Title: Brain-specific angiogenesis inhibitor 1 is expressed in the Myo/Nog cell lineage

doi: 10.1371/journal.pone.0234792

Figure Lengend Snippet: An alanine-scan library of BAI1 was constructed. The G8 and reference 5D6 mAbs were screened for binding to each individual BAI1 variant. A. Identification of candidate critical clones for G8 mAb binding was determined in duplicate by high throughput flow cytometry. For each point, background fluorescence was subtracted from the raw data that were then normalized to G8 reactivity with wild type BAI1. For each mutant clone, the mean binding value was plotted as a function of expression represented by control antibody reactivity. Candidate critical clones (red circles) were identified with a threshold (dashed line) of <40% binding of the G8 mAb and >50% of binding of the control 5D6 mAb to wild type BAI1. B. Identification of validated critical residues for G8 mAb binding. Candidate critical residues for G8 were rescreened in quadruplicate and the mean binding reactivities obtained. Validated critical residues for G8 binding (outlined in red) were residues whose mutations that resulted in <20% of wild type binding but positive (>70% binding) for binding of the 5D6 control mAb. Additional validated secondary residues (outlined in blue) were identified that did not meet the threshold guidelines but whose decreased binding activity and proximity to critical residues suggested that they may be part of the antibody epitope. C. Visualization of validated critical residues for G8 mAb binding. Critical residues (red spheres) and secondary residues (blue spheres) which may also contribute to binding were visualized on two Phyre-generated model structures that were combined to model BAI1 residues 262–934. The G8 critical residues were visualized on a model of the BAI1 thrombospondin repeat domains based on the structure of properdin (PDB ID# 1WOR; .

Article Snippet: Both G8 and anti-BAI1 mAb from R&D Systems (R&D BAI1) bound to a substrate of purified BAI1 protein containing amino acids 31–879 located within the extracellular domain ( ).

Techniques: Construct, Binding Assay, Variant Assay, Clone Assay, High Throughput Screening Assay, Flow Cytometry, Fluorescence, Mutagenesis, Expressing, Control, Activity Assay, Generated

Journal: PLoS ONE

Article Title: Brain-specific angiogenesis inhibitor 1 is expressed in the Myo/Nog cell lineage

doi: 10.1371/journal.pone.0234792

Figure Lengend Snippet: Localization of antibodies to BAI1, Noggin and MyoD in the skin, eyes and brain. Human lens tissue and tissue sections of human tattooed skin, rabbit anterior cavity, rat retina and mouse brain were double labeled with the G8 and R&D BAI1 mAbs and antibodies to Noggin and MyoD. The total numbers of double labeled cells were counted in a subset of tissue sections. The total numbers of single labeled cells were quantified in all sections with the exception of MyoD-/R&D BAI1 mAb+ and MyoD-/G8- cells that were counted in a subset of skin and anterior cavity sections. The numbers of tissues and sections scored are indicated in parenthesis. The results are the mean ± standard deviation. The R&D BAI1 mAb co-localized with G8 and Noggin with rare exception. BAI1+ cells without MyoD were present in the skin and anterior cavity.

Article Snippet: Both G8 and anti-BAI1 mAb from R&D Systems (R&D BAI1) bound to a substrate of purified BAI1 protein containing amino acids 31–879 located within the extracellular domain ( ).

Techniques: Labeling, Standard Deviation

Journal: PLoS ONE

Article Title: Brain-specific angiogenesis inhibitor 1 is expressed in the Myo/Nog cell lineage

doi: 10.1371/journal.pone.0234792

Figure Lengend Snippet: Tissue sections of human tattooed skin (A-C), human anterior lens tissue (D-F), rabbit eyes (G-I) and rat retina (J-L) were double labeled with the G8 (red) and BAI1 (green) mAbs. Nuclei were stained with Hoechst dye (blue). Unmerged images are shown in A, B, D, E and the insets in G-I and K and L. Overlap of red and green appear yellow in triple merged images in C, F, G-I, K and L. A merge of fluorescence and DIC is shown in the inset of C. The G8 and BAI1 mAbs labeled the same cells in the skin, human lens, rabbit lens (G), ciliary body (H) and cornea (I), and the inner plexiform and inner nuclear layers of the mouse retina (K and L). Minimal background fluorescence was observed in the anti-IgM and anti-IgG secondary antibody controls for the skin (M), human lens tissue (N), rabbit lens (O) and mouse retina P and Q. Bar = 9 μM in A-I and K-Q and 54 μM in J.

Article Snippet: Both G8 and anti-BAI1 mAb from R&D Systems (R&D BAI1) bound to a substrate of purified BAI1 protein containing amino acids 31–879 located within the extracellular domain ( ).

Techniques: Labeling, Staining, Fluorescence

Journal: PLoS ONE

Article Title: Brain-specific angiogenesis inhibitor 1 is expressed in the Myo/Nog cell lineage

doi: 10.1371/journal.pone.0234792

Figure Lengend Snippet: Tissue sections of human tattooed skin (A and B), human anterior lens tissue (C) and rabbit anterior cavity (D-I) were double labeled with the R&D BAI1 mAb and antibodies to noggin or MyoD. The colors of the fluorescent secondary antibodies are indicated in each photograph. Overlap of red and green appears yellow in merged images. Nuclei were stained with Hoechst dye. Double labeled cells were present in the skin (A and B), anterior human lens tissue (C) and the rabbit lens (D and G), ciliary body (E and H) and cornea (F and I). Bar = 9 μM.

Article Snippet: Both G8 and anti-BAI1 mAb from R&D Systems (R&D BAI1) bound to a substrate of purified BAI1 protein containing amino acids 31–879 located within the extracellular domain ( ).

Techniques: Labeling, Staining

Journal: PLoS ONE

Article Title: Brain-specific angiogenesis inhibitor 1 is expressed in the Myo/Nog cell lineage

doi: 10.1371/journal.pone.0234792

Figure Lengend Snippet: Tissue sections from the day-10 mouse brain were stained with hematoxylin and eosin (sagittal sections A and D; coronal sections B and C) or double labeled with the G8 and the R&D BAI1 mAbs, or G8 and antibodies to Iba1, NeuN or GFAP. The areas within the boxes of the H&E stained sections are shown at high magnification in the fluorescence photomicrographs. The colors of the fluorescent secondary antibodies are indicated in the unmerged photographs (E, F, H and I). Nuclei were stained with Hoechst dye. Overlap of red and green, when present, appears yellow in merged images (G, J, K, L and M). The G8 and BAI1 mAbs labeled the same subpopulation of cells in the hippocampal formation (box in A; E-G). The Noggin and BAI1 antibodies also bound to the same cells in the glomerular layer of the olfactory bulb (box in B; H-J). G8 did not co-localize with Iba1 (K, from box in A), NeuN (L from lower box in C) or GFAP (M from box in D). Minimal fluorescence was observed with the anti-IgM and anti-IgG (inset in G) or the anti-goat and anti-IgG (inset in J) secondary antibodies. Bar = 270 μM in A-D and 9 μM in E-M.

Article Snippet: Both G8 and anti-BAI1 mAb from R&D Systems (R&D BAI1) bound to a substrate of purified BAI1 protein containing amino acids 31–879 located within the extracellular domain ( ).

Techniques: Staining, Labeling, Fluorescence

Journal: PLoS ONE

Article Title: Brain-specific angiogenesis inhibitor 1 is expressed in the Myo/Nog cell lineage

doi: 10.1371/journal.pone.0234792

Figure Lengend Snippet: Tissue sources and numbers of tissue and tissue sections screened by immunofluorescence localization of antibodies.

Article Snippet: The G8 IgM mAb and BAI1 IgG mAb (MAB4969, R&D Systems, Minneapolis, MN) were added in 100 μl of phosphate buffered saline (Applied Biosciences, ThermoFisher Scientific) at concentrations of 0.18–1.8 μg/well and 0.08–0.2 μg/100 μl/well, respectively.

Techniques: Immunofluorescence

Journal: PLoS ONE

Article Title: Brain-specific angiogenesis inhibitor 1 is expressed in the Myo/Nog cell lineage

doi: 10.1371/journal.pone.0234792

Figure Lengend Snippet: The G8 mAb was used to screen clones of HEK-239T cells expressing a single human membrane protein. G8 binding was detected by flow cytometry. A. Binding values were normalized and transformed to give a single numerical value for binding of the G8 mAb against each target protein (normalized target binding). Non-specific fluorescence was determined to be any value below three standard deviations above noise. G8 binding above background occurred with the clone expressing BAI1. B. Validation of binding of the G8 mAb to BAI1 or vector alone was carried out with HEK-293T cells transfected with the plasmid construct expressing target using a serial dilution of mAb. C. Binding of different concentrations of the G8 mAb was calculated as the signal to background ratio of fluorescence from the BAI1 expressing clone to empty vector clone.

Article Snippet: The G8 IgM mAb and BAI1 IgG mAb (MAB4969, R&D Systems, Minneapolis, MN) were added in 100 μl of phosphate buffered saline (Applied Biosciences, ThermoFisher Scientific) at concentrations of 0.18–1.8 μg/well and 0.08–0.2 μg/100 μl/well, respectively.

Techniques: Clone Assay, Expressing, Membrane, Binding Assay, Flow Cytometry, Transformation Assay, Fluorescence, Biomarker Discovery, Plasmid Preparation, Transfection, Construct, Serial Dilution

Journal: PLoS ONE

Article Title: Brain-specific angiogenesis inhibitor 1 is expressed in the Myo/Nog cell lineage

doi: 10.1371/journal.pone.0234792

Figure Lengend Snippet: Plates were coated with recombinant human BAI1 protein produced by CHO cells. Binding of the G8 IgM mAb and R&D BAI1 IgG mAb (a-BAI) was detected with anti-mouse IgM and IgG affinity purified F(ab’)2 secondary antibodies, respectively, conjugated with horseradish peroxidase. Both mAbs bound to BAI1 protein.

Article Snippet: The G8 IgM mAb and BAI1 IgG mAb (MAB4969, R&D Systems, Minneapolis, MN) were added in 100 μl of phosphate buffered saline (Applied Biosciences, ThermoFisher Scientific) at concentrations of 0.18–1.8 μg/well and 0.08–0.2 μg/100 μl/well, respectively.

Techniques: Recombinant, Produced, Binding Assay, Affinity Purification

Journal: PLoS ONE

Article Title: Brain-specific angiogenesis inhibitor 1 is expressed in the Myo/Nog cell lineage

doi: 10.1371/journal.pone.0234792

Figure Lengend Snippet: An alanine-scan library of BAI1 was constructed. The G8 and reference 5D6 mAbs were screened for binding to each individual BAI1 variant. A. Identification of candidate critical clones for G8 mAb binding was determined in duplicate by high throughput flow cytometry. For each point, background fluorescence was subtracted from the raw data that were then normalized to G8 reactivity with wild type BAI1. For each mutant clone, the mean binding value was plotted as a function of expression represented by control antibody reactivity. Candidate critical clones (red circles) were identified with a threshold (dashed line) of <40% binding of the G8 mAb and >50% of binding of the control 5D6 mAb to wild type BAI1. B. Identification of validated critical residues for G8 mAb binding. Candidate critical residues for G8 were rescreened in quadruplicate and the mean binding reactivities obtained. Validated critical residues for G8 binding (outlined in red) were residues whose mutations that resulted in <20% of wild type binding but positive (>70% binding) for binding of the 5D6 control mAb. Additional validated secondary residues (outlined in blue) were identified that did not meet the threshold guidelines but whose decreased binding activity and proximity to critical residues suggested that they may be part of the antibody epitope. C. Visualization of validated critical residues for G8 mAb binding. Critical residues (red spheres) and secondary residues (blue spheres) which may also contribute to binding were visualized on two Phyre-generated model structures that were combined to model BAI1 residues 262–934. The G8 critical residues were visualized on a model of the BAI1 thrombospondin repeat domains based on the structure of properdin (PDB ID# 1WOR; .

Article Snippet: The G8 IgM mAb and BAI1 IgG mAb (MAB4969, R&D Systems, Minneapolis, MN) were added in 100 μl of phosphate buffered saline (Applied Biosciences, ThermoFisher Scientific) at concentrations of 0.18–1.8 μg/well and 0.08–0.2 μg/100 μl/well, respectively.

Techniques: Construct, Binding Assay, Variant Assay, Clone Assay, High Throughput Screening Assay, Flow Cytometry, Fluorescence, Mutagenesis, Expressing, Control, Activity Assay, Generated

Journal: PLoS ONE

Article Title: Brain-specific angiogenesis inhibitor 1 is expressed in the Myo/Nog cell lineage

doi: 10.1371/journal.pone.0234792

Figure Lengend Snippet: Localization of antibodies to BAI1, Noggin and MyoD in the skin, eyes and brain. Human lens tissue and tissue sections of human tattooed skin, rabbit anterior cavity, rat retina and mouse brain were double labeled with the G8 and R&D BAI1 mAbs and antibodies to Noggin and MyoD. The total numbers of double labeled cells were counted in a subset of tissue sections. The total numbers of single labeled cells were quantified in all sections with the exception of MyoD-/R&D BAI1 mAb+ and MyoD-/G8- cells that were counted in a subset of skin and anterior cavity sections. The numbers of tissues and sections scored are indicated in parenthesis. The results are the mean ± standard deviation. The R&D BAI1 mAb co-localized with G8 and Noggin with rare exception. BAI1+ cells without MyoD were present in the skin and anterior cavity.

Article Snippet: The G8 IgM mAb and BAI1 IgG mAb (MAB4969, R&D Systems, Minneapolis, MN) were added in 100 μl of phosphate buffered saline (Applied Biosciences, ThermoFisher Scientific) at concentrations of 0.18–1.8 μg/well and 0.08–0.2 μg/100 μl/well, respectively.

Techniques: Labeling, Standard Deviation

Journal: PLoS ONE

Article Title: Brain-specific angiogenesis inhibitor 1 is expressed in the Myo/Nog cell lineage

doi: 10.1371/journal.pone.0234792

Figure Lengend Snippet: Tissue sections of human tattooed skin (A-C), human anterior lens tissue (D-F), rabbit eyes (G-I) and rat retina (J-L) were double labeled with the G8 (red) and BAI1 (green) mAbs. Nuclei were stained with Hoechst dye (blue). Unmerged images are shown in A, B, D, E and the insets in G-I and K and L. Overlap of red and green appear yellow in triple merged images in C, F, G-I, K and L. A merge of fluorescence and DIC is shown in the inset of C. The G8 and BAI1 mAbs labeled the same cells in the skin, human lens, rabbit lens (G), ciliary body (H) and cornea (I), and the inner plexiform and inner nuclear layers of the mouse retina (K and L). Minimal background fluorescence was observed in the anti-IgM and anti-IgG secondary antibody controls for the skin (M), human lens tissue (N), rabbit lens (O) and mouse retina P and Q. Bar = 9 μM in A-I and K-Q and 54 μM in J.

Article Snippet: The G8 IgM mAb and BAI1 IgG mAb (MAB4969, R&D Systems, Minneapolis, MN) were added in 100 μl of phosphate buffered saline (Applied Biosciences, ThermoFisher Scientific) at concentrations of 0.18–1.8 μg/well and 0.08–0.2 μg/100 μl/well, respectively.

Techniques: Labeling, Staining, Fluorescence

Journal: PLoS ONE

Article Title: Brain-specific angiogenesis inhibitor 1 is expressed in the Myo/Nog cell lineage

doi: 10.1371/journal.pone.0234792

Figure Lengend Snippet: Tissue sections of human tattooed skin (A and B), human anterior lens tissue (C) and rabbit anterior cavity (D-I) were double labeled with the R&D BAI1 mAb and antibodies to noggin or MyoD. The colors of the fluorescent secondary antibodies are indicated in each photograph. Overlap of red and green appears yellow in merged images. Nuclei were stained with Hoechst dye. Double labeled cells were present in the skin (A and B), anterior human lens tissue (C) and the rabbit lens (D and G), ciliary body (E and H) and cornea (F and I). Bar = 9 μM.

Article Snippet: The G8 IgM mAb and BAI1 IgG mAb (MAB4969, R&D Systems, Minneapolis, MN) were added in 100 μl of phosphate buffered saline (Applied Biosciences, ThermoFisher Scientific) at concentrations of 0.18–1.8 μg/well and 0.08–0.2 μg/100 μl/well, respectively.

Techniques: Labeling, Staining

Journal: PLoS ONE

Article Title: Brain-specific angiogenesis inhibitor 1 is expressed in the Myo/Nog cell lineage

doi: 10.1371/journal.pone.0234792

Figure Lengend Snippet: Tissue sections from the day-10 mouse brain were stained with hematoxylin and eosin (sagittal sections A and D; coronal sections B and C) or double labeled with the G8 and the R&D BAI1 mAbs, or G8 and antibodies to Iba1, NeuN or GFAP. The areas within the boxes of the H&E stained sections are shown at high magnification in the fluorescence photomicrographs. The colors of the fluorescent secondary antibodies are indicated in the unmerged photographs (E, F, H and I). Nuclei were stained with Hoechst dye. Overlap of red and green, when present, appears yellow in merged images (G, J, K, L and M). The G8 and BAI1 mAbs labeled the same subpopulation of cells in the hippocampal formation (box in A; E-G). The Noggin and BAI1 antibodies also bound to the same cells in the glomerular layer of the olfactory bulb (box in B; H-J). G8 did not co-localize with Iba1 (K, from box in A), NeuN (L from lower box in C) or GFAP (M from box in D). Minimal fluorescence was observed with the anti-IgM and anti-IgG (inset in G) or the anti-goat and anti-IgG (inset in J) secondary antibodies. Bar = 270 μM in A-D and 9 μM in E-M.

Article Snippet: The G8 IgM mAb and BAI1 IgG mAb (MAB4969, R&D Systems, Minneapolis, MN) were added in 100 μl of phosphate buffered saline (Applied Biosciences, ThermoFisher Scientific) at concentrations of 0.18–1.8 μg/well and 0.08–0.2 μg/100 μl/well, respectively.

Techniques: Staining, Labeling, Fluorescence