Journal: bioRxiv

Article Title: Uncovering the electrical synapse proteome in retinal neurons via in vivo proximity labeling

doi: 10.1101/2024.11.26.625481

Figure Lengend Snippet: (A) Proteins implicated in membrane trafficking SJ2BP and Syt4 colocalize with Cx36 in AII amacrine cells. (B) Several cytoskeleton associated proteins and regulators such as MAP6, DOCK7 or GPrin1 colocalize with Cx36 in AII cell dendrites. (C) The adhesion molecule BAI1 was often colocalized with Cx36 in AII cell dendrites. (D) Often components of chemical synapses such as SHANK2 and Glur2-3 were found in the periphery of gap junction plaques in AII amacrine cells. Scale: 10 µm. Magnified inset: 1 µm.

Article Snippet: The following primary antibodies were used: Cx36, 1:250 (Clone: 8F6, MAB3045, Millipore); Cx36, 1:250 (Clone: 1E5H5, 37-4600, Thermofisher); GFP, 1:250 (A10262, Thermofisher); ZO-1, 1:250 (Clone: ZO1-1A12, 33-9100, Thermofisher); ZO-2, 1:250 (71-1400, Thermofisher); CGN, 1:250 (PA5-5561, Thermofisher); SIPA1L3, 1:100 (30544-1-AP, Proteintech); EPS15L1, 1:250 (PA5-65940, Thermofisher); HIPR1, 1:250 (AB9882,Sigma); GluR2-3, 1:200 (07-598, Sigma); Gprin1, 1:100 (13771-1-AP, Proteintech); DOCK7, 1:100 (13000-1-AP, Proteintech); MAP6, 1:100 (NBP2-14220, Novus Biologicals); Synaptotagmin4, 1:100 (105 143, Synaptic Systems); SJ2BP, 1:100 (15666-1-AP, Proteintech); BAI1, 1:100 (NB110-81586, Novus Biologicals); SHANK2, 1:500 (Synaptic Systems, 162204); SCGN, 1:250 (CSB-PA020821LA01HU, ARP); SCGN, 1:500 (RD184120100, Biovendor); Vglut1, 1:500 (AB5905, Sigma); ITNS1, 1:100-250 (PA5-115432, Thermofisher).

Techniques: Membrane

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