rabbit anti mouse ionized calcium binding adaptor molecule 1  (Novus Biologicals)

Journal: Stem Cell Research & Therapy

Article Title: Spatiotemporal evolution of early innate immune responses triggered by neural stem cell grafting

doi: 10.1186/scrt147

Figure Lengend Snippet: Histological analysis of neural stem cell (NSC) graft survival and endogenous glial cell responses . First row, NSC-Luciferase/enhanced fluorescent green protein (Luc/eGFP) graft survival. Direct eGFP fluorescence (green) combined with TOPRO3 staining (false colour representation in blue) at day 0, 1, 3, 5, 7 and 14 post-implantation. Representative images were chosen from multiple stained slides (n = 6 to 9 for eGFP/TOPRO3 combination) per mouse analysed at each time point. The provided scale bars indicate 200 μm. Second row, cellular hypoxia. Direct eGFP fluorescence (green) combined with Hypoxyprobe-1staining (red) at day 0, 1, 3, 5, 7 and 14 post-implantation. Representative images were chosen from two to five mice analysed at each time point. The provided scale bars indicate 50 μm. Third, fourth and fifth row, endogenous glial cell behaviour. Direct eGFP fluorescence (green) combined with TOPRO3 staining (false colour representation in blue) and combined with immunofluorescence staining for ionized calcium binding adaptor molecule 1 (Iba1) (red, fourth row), S100 calcium binding protein B (S100B) (red, fifth row) or glial fibrillary acidic protein (GFAP) (red, sixth row) at day 0, 1, 3, 5, 7 and 14 post-implantation. Representative images were chosen from multiple stained slides (n = 3 for eGFP/TOPRO3/Iba1 combination, n = 3 for eGFP/TOPRO3/S100B combination and n = 1 for eGFP/TOPRO3/GFAP) per mouse analysed at each time point (n = 4/5). The provided scale bars indicate 50 μm for Iba1 and S100B images and 200 μm for GFAP images. Sixth row, graft site remodelling. Direct eGFP fluorescence (green) combined with myelin base protein (MBP) staining (red) at day 0, 1, 3, 5, 7 and 14 post-implantation. Representative images were chosen from multiple mice analysed at each time point (n = 2). The provided scale bars indicate 200 μm.

Article Snippet: For further immunofluorescence analysis of tissue sections, antibody staining was performed as previously described [ , , ], using the following antibodies: (i) a rabbit anti-mouse ionized calcium binding adaptor molecule 1 (Iba1) antibody (1/200) (Wako chemicals, Osaka, Japan; 019-19714) and (ii) a rabbit anti-pimonidazole (Hypoxyprobe-1) antibody (1/200) (HPI Inc., Burlington, MA, USA; Pab2627), all in combination with a secondary donkey anti-rabbit AlexaFluor 555 antibody (1/500) (Life Technologies, Carlsbad, CA, USA; A31572), (iv) a rabbit anti-mouse S100B antibody (1/200) (Abcam, Cambridge, UK; 52642) in combination with a secondary donkey anti-rabbit AlexaFluor 555 antibody (1/1000) (Life Technologies, Carlsbad, CA, USA; A31572), (v) a mouse anti-mouse GFAP antibody (1/400) (Millipore, Billerica, MA, USA; MAB360) in combination with a secondary goat anti-mouse AlexaFluor 555 (1/1000) (Life Technologies, Carlsbad, CA, USA; A21127), and (vi) a chicken anti-mouse MBP antibody (1/200) (Millipore, Billerica, MA, USA; AB9348) in combination with a secondary donkey anti-chicken DyLight 549 (Jackson Immunoresearch, Suffolk, UK; 703-506-155).

Techniques: Luciferase, Fluorescence, Staining, Immunofluorescence, Binding Assay

Journal: Stem Cell Research & Therapy

Article Title: Spatiotemporal evolution of early innate immune responses triggered by neural stem cell grafting

doi: 10.1186/scrt147

Figure Lengend Snippet: In vivo cell graft and glial cell behaviour - quantitative analysis . (a) Survival of grafted neural stem cell-Luciferase/enhanced fluorescent green protein (NSC-Luc/eGFP). Presented data are the estimated total number of eGFP-expressing NSC-Luc/eGFP detected within the implant site for each mouse analysed (n = 5 for day 0, n = 5 for day 1, n = 4 for day 3, n = 4 for day 5, n = 4 for day 7 and n = 4 for day 14). The red line indicates the average total number of eGFP-expressing NSC-Luc/eGFP at each time point analysed. The inset graph represents the mean % (± standard error of the mean (SEM)) of NSC-Luc/eGFP graft survival at each time point analysed from day 1 post-implantation. Significant differences are described in the results section. (b-e) Cellular density of ionized calcium binding adaptor molecule 1 (Iba1) pos microglia and S100 calcium binding protein B (S100B) pos astrocytes within the implant site and within the implant border. Presented data are the ln(x)-transformed values of estimated total density of microglia and astrocytes within the implant border for each mouse analysed (n = 4 for day 0, n = 5 for day 1, n = 4 for day 3, n = 5 for day 5, n = 4 for day 7 and n = 4 for day 14). The red line indicates the average total cellular density at each time point analysed. Significant differences are described in the results section. (f) Astrogliosis within the implant site and implant border (both areas are combined here). Presented data indicate the average degree of glial fibrillary acidic protein (GFAP) pos astrogliosis (based on image coverage after GFAP staining) for each mouse analyses (n = 4 for day 0, n = 5 for day 1, n = 4 for day 3, n = 5 for day 5, n = 4 for day 7 and n = 4 for day 14). The red line indicates the average degree of astrogliosis at each time point analysed. Significant differences are described in the results section.

Article Snippet: For further immunofluorescence analysis of tissue sections, antibody staining was performed as previously described [ , , ], using the following antibodies: (i) a rabbit anti-mouse ionized calcium binding adaptor molecule 1 (Iba1) antibody (1/200) (Wako chemicals, Osaka, Japan; 019-19714) and (ii) a rabbit anti-pimonidazole (Hypoxyprobe-1) antibody (1/200) (HPI Inc., Burlington, MA, USA; Pab2627), all in combination with a secondary donkey anti-rabbit AlexaFluor 555 antibody (1/500) (Life Technologies, Carlsbad, CA, USA; A31572), (iv) a rabbit anti-mouse S100B antibody (1/200) (Abcam, Cambridge, UK; 52642) in combination with a secondary donkey anti-rabbit AlexaFluor 555 antibody (1/1000) (Life Technologies, Carlsbad, CA, USA; A31572), (v) a mouse anti-mouse GFAP antibody (1/400) (Millipore, Billerica, MA, USA; MAB360) in combination with a secondary goat anti-mouse AlexaFluor 555 (1/1000) (Life Technologies, Carlsbad, CA, USA; A21127), and (vi) a chicken anti-mouse MBP antibody (1/200) (Millipore, Billerica, MA, USA; AB9348) in combination with a secondary donkey anti-chicken DyLight 549 (Jackson Immunoresearch, Suffolk, UK; 703-506-155).

Techniques: In Vivo, Luciferase, Expressing, Binding Assay, Transformation Assay, Staining

Journal: Journal of Ocular Pharmacology and Therapeutics

Article Title: Amount of Mononuclear Phagocyte Infiltrate Does Not Predict Area of Experimental Choroidal Neovascularization (CNV)

doi: 10.1089/jop.2017.0131

Figure Lengend Snippet: Laser induced injury to mouse eyes induces Iba-1 + cellular infiltration into the subretinal space. (A) 20× confocal image of overlapping confluent Iba-1 + cells ( green ) on top of a CNV lesion labeled with Fluorescein Concanavalin A in a mouse PEC collected 7 days after laser application. (B) 5× image of a mouse PEC 7 days after laser injury applied in 3 regions. (C) Same image as (B) , with individual Iba-1 + cells peripheral to the CNV highlighted in purple by MATLAB analysis software. ( D ) Bar graph of number of Iba-1 + cells in the subretinal space of RPE-choroid flat mounts, peripheral to the CNV at day 3 (D3) and day 7 (D7) compared with naive nonlasered PEC ± SEM. Day 7 lasered mice exhibited the highest cellular infiltrate compared to the nonlasered mice. Iba1 + cell counts were analyzed in nonlasered mice collected either 3 or 7 days after anesthesia. As microglia counts were similar in nonlasered mice at both time points, these nonlasered mice were combined into 1 group for comparison to the lasered mice. Data presented are the number of peripheral microglia in 1 PEC sample ± SEM and are combined from 8 individual studies, n = 66–97 data points for each condition. Scale bar figure (A) 200 μm, (B) 500 μm. ONH, optic nerve head ( arrow ). CNV, choroidal neovascularization; PEC, posterior eye cup; RPE, retinal pigment epithelium.

Article Snippet: The tissues were blocked in PBS with 1% bovine serum albumin (BSA) (EMD Millipore, Billerica, MA) for 1–2 h. PECs were incubated overnight with rabbit anti-mouse ionized calcium binding adaptor molecule-1 (Iba1, 019-19741; Wako Chemicals, Cambridge, MA) 1:1,000 to label microglia and/or macrophages (MNP cells found in ocular tissues) and biotin-Ly-6G/C, also known as Gr1 (108403; BioLegend, San Diego, CA), 1:1,000 in staining buffer to label neutrophils.

Techniques: Labeling, Software

Journal: Journal of Ocular Pharmacology and Therapeutics

Article Title: Amount of Mononuclear Phagocyte Infiltrate Does Not Predict Area of Experimental Choroidal Neovascularization (CNV)

doi: 10.1089/jop.2017.0131

Figure Lengend Snippet: Systemic administration of PAM increased the number of Iba-1 + cells in the subretinal space, over the CNV lesion, and increased neutrophil infiltration into the CNV lesion after laser injury. Representative 5 × image of a mouse PEC 7 days after laser injury in mice injected with (A) Vehicle or (B) PAM. (C) Bar graph and (D) Dot plot from a study demonstrating the number of Iba1 + cells in mice injected with PAM ( n = 8–10 eyes of 4–5 mice per condition, P < 0.05). (E) Bar graph and (F) Dot plot of mean intensity of Iba1 + label on CNV lesions ( n = 8–10 eyes, 20–29 CNV analyzed per condition, P < 0.01). 20 × images of CNV labeled with GR1 from mice treated with PBS (G) or PAM (H) Collected 3 days after laser. (I) Bar graph and (J) Dot plot of number of GR1 + neutrophils per CNV lesion ( P < 0.001 at day 3, > 0.05 at day 7). Scale bar (A) and (B) 500 μm and (G) and (H) 200 μm. Statistics performed with an unpaired t -test. ONH, optic nerve head ( arrow ).

Article Snippet: The tissues were blocked in PBS with 1% bovine serum albumin (BSA) (EMD Millipore, Billerica, MA) for 1–2 h. PECs were incubated overnight with rabbit anti-mouse ionized calcium binding adaptor molecule-1 (Iba1, 019-19741; Wako Chemicals, Cambridge, MA) 1:1,000 to label microglia and/or macrophages (MNP cells found in ocular tissues) and biotin-Ly-6G/C, also known as Gr1 (108403; BioLegend, San Diego, CA), 1:1,000 in staining buffer to label neutrophils.

Techniques: Injection, Labeling

Journal: Journal of Ocular Pharmacology and Therapeutics

Article Title: Amount of Mononuclear Phagocyte Infiltrate Does Not Predict Area of Experimental Choroidal Neovascularization (CNV)

doi: 10.1089/jop.2017.0131

Figure Lengend Snippet: Iba1 + cell infiltration after laser is not reduced by anti-VEGF therapy. Representative 5 × images of mouse PEC collected 7 days after laser labeled with Iba1 + treated with (A) PBS or (B) 4G3. CNV lesions are circled. (C) Bar graph and (D) Dot plot of mean number of discrete Iba1 + cells peripheral to CNV lesions ( n = 5 mice, 9–10 data points per condition). (E) Bar graph and (F) Dot plot of mean integrated intensity of Iba1 + label in ROI centered on CNV ( n = 5 mice, 24–26 data points included in analysis/condition, P > 0.05). Scale bar = 500 μm. ONH, optic nerve head ( arrow ). AU, arbitrary units; ROI, region of interest.

Article Snippet: The tissues were blocked in PBS with 1% bovine serum albumin (BSA) (EMD Millipore, Billerica, MA) for 1–2 h. PECs were incubated overnight with rabbit anti-mouse ionized calcium binding adaptor molecule-1 (Iba1, 019-19741; Wako Chemicals, Cambridge, MA) 1:1,000 to label microglia and/or macrophages (MNP cells found in ocular tissues) and biotin-Ly-6G/C, also known as Gr1 (108403; BioLegend, San Diego, CA), 1:1,000 in staining buffer to label neutrophils.

Techniques: Labeling

Journal: Frontiers in Cellular Neuroscience

Article Title: Globular Adiponectin Limits Microglia Pro-Inflammatory Phenotype through an AdipoR1/NF-κB Signaling Pathway

doi: 10.3389/fncel.2017.00352

Figure Lengend Snippet: Adiponectin deficiency leads to microglia hyper-responsiveness toward LPS. (A) Representative images of Iba-1-immunoreactive microglia (green fluorescence) in hippocampus (left panels) and hypothalamus (right panels) from Wt (upper panels) and ApN −/− (lower panels) mouse brains, 24 h after LPS challenge. Nuclei are stained with Hoechst fluorescent dye (blue fluorescence). All images were taken under the same parameters. (B) Quantification of Iba-1 staining using fluorescence intensity values to quantify Iba-1 levels. (C) Schematic representation of the protocol. 24 h after LPS ip injection, IL-1β, IL-6, and TNFα proteins (D) or mRNAs (E) were quantified by CBA or qRT-PCR, respectively, from microglia sorted from brain cell suspensions of Wt (white bars) or ApN −/− mice (black bars) cultured for 15 h. (D) Data represent the mean concentration ± SEM, n = 4. (E) Data represent the mean expression levels ± s expressed as fold change compared to LPS-treated Wt mouse microglia, n = 4. Mann & Whitney for comparison between groups, * p < 0.05, vs. control.

Article Snippet: Sections were washed and incubated with a rabbit anti-mouse ionized calcium binding adaptor molecule 1 (Iba-1) antibody (Wako Chemicals, USA), overnight at 4°C, followed by incubation with the appropriate fluorescent-conjugated secondary antibody (Invitrogen).

Techniques: Fluorescence, Staining, Injection, Quantitative RT-PCR, Cell Culture, Concentration Assay, Expressing, MANN-WHITNEY