Immunohistochemistry was performed as described previously37 (link). Briefly, 10 μm thick cryosections were fixed in 4% paraformaldehyde for 20 min at room temperature and then incubated in 0.1% Triton X100/phosphate-buffered saline (PBS) for 20 min at 37 °C followed by incubation in 3% bovine serum albumin/PBS for 1 h at room temperature. The cryosections were then incubated with the following primary antibodies for 1 day at 4 °C: rabbit polyclonal antibody against α7-nAChR (sc-5544, 1:20; Santa Cruz Biotechnology), rabbit polyclonal antibody against GAD65/GAD67 (ab11070, 1:200; Abcam), rabbit polyclonal antibody against GABA (A2052, 1:100; Sigma, St. Louis, MO, USA), and goat polyclonal antibody against Brn3a (sc-31984, 1:40; Santa Cruz Biotechnology). The secondary antibodies (all from Invitrogen-Molecular Probes, Eugene, OR, USA) were Alexa Fluor 555-conjugated donkey anti-rabbit IgG antibody (A31572, 1:1,000), Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody (A11070, 1:500), and Alexa Fluor 488-conjugated donkey anti-goat IgG antibody (A11055, 1:500). The sections were finally counterstained with the nucleic acid stain Hoechst 33258 (H3569, 1:2,000; Invitrogen-Molecular Probes) in PBS and imaged using a laser scanning confocal microscope (TCS SP8; Leica Microsystems, Heidelberg, Germany).
Sc 31984
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-31984 is a laboratory equipment product offered by Santa Cruz Biotechnology. It is designed to facilitate specific research applications. The core function of this product is to provide a tool for researchers to conduct their investigations. Further details on the intended use or specifications of Sc-31984 are not available.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti brn3a, by Santa Cruz Biotechnology (3 mentions)
Fv1000, by Olympus (2 mentions)
Ab11070, by Abcam (1 mentions)
A2052, by Merck Group (1 mentions)
A11070, by Thermo Fisher Scientific (1 mentions)
A11055, by Thermo Fisher Scientific (1 mentions)
Hoechst 33258, by Thermo Fisher Scientific (1 mentions)
Tcs sp8, by Leica Microsystems (1 mentions)
Sc 14363, by Santa Cruz Biotechnology (1 mentions)
Anti brn3a, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 594, by Jackson ImmunoResearch (1 mentions)
Brn3a, by Santa Cruz Biotechnology (1 mentions)
C57bl 6j, by Charles River Laboratories (1 mentions)
E3 system, by Diagnosys (1 mentions)
Full field ganzfeld sphere, by Diagnosys (1 mentions)
Confocal microscope, by Leica Microsystems (1 mentions)
Ab87117, by Abcam (1 mentions)
Mabn92, by Merck Group (1 mentions)
Prolong gold antifade, by Thermo Fisher Scientific (1 mentions)
Fluorescence microscope, by Keyence (1 mentions)
11 protocols using sc 31984
1
Immunohistochemistry of Neuronal Markers
2
Retinal Immunohistochemistry with Opsin and Brn3a
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Primary antibodies: S-opsin∶goat anti-OPN1SW, 1∶1000 (sc-14363, Santa-Cruz Biotechnologies, Heidelberg, Germany), L-opsin∶rabbit anti-opsin red/green, 1∶1200 (ab5405, Millipore Ibérica, Madrid, Spain), Brn3a∶goat anti-Brn3a, 1∶750 (sc-31984, Santa-Cruz Biotechnologies, Heidelberg, Germany).
Secondary antibodies: donkey anti-goat Alexa Fluor 488, donkey anti-rabbit Alexa Fluor 488 and donkey anti-rabbit Alexa Fluor 594 (Jackson ImmunoResearch, Suffolk, UK), all used at 1∶500.
Secondary antibodies: donkey anti-goat Alexa Fluor 488, donkey anti-rabbit Alexa Fluor 488 and donkey anti-rabbit Alexa Fluor 594 (Jackson ImmunoResearch, Suffolk, UK), all used at 1∶500.
3
Intravitreal Injection and Retinal Ganglion Cell Quantification in Mice
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9- to 12-week-old male and female C57BL/6J (Charles River, UK, RRID:IMSR_JAX:000664 ), or Sarm1-/- (RRID:MGI:3765957 ) mice were intravitreally injected with either 2 μL vacor, DMSO (vehicle) or PBS. Injections were performed as previously described (Osborne et al., 2018 (link)). ERG responses were recorded simultaneously from both eyes using an Espion E3 system with full-field Ganzfeld sphere (Diagnosys, Cambridge, UK). Animals were dark-adapted overnight, and ERG recordings performed under low level, red light illumination. Peak RGC responses (pSTR) were recorded between 70–110 ms after a light exposure of –4.73 log cd.s.m–2, B wave peaks between 80–120 ms, at –1.90 log cd.s.m–2, and A wave troughs within the first 20 ms at 1.29 log cd.s.m–2. Following culling via exposure to CO2, eyes were post-fixed for 24 hr in 4 % PFA before retinal flatmounts were prepared as previously described (Osborne et al., 2018 (link)). Briefly, retinas were excised from the eye cup, flattened and stained with the RGC specific nuclei marker BRN3A (Santa Cruz, sc-31984, RRID:AB_2167511 , 1:200). Images were captured, blindly, from eight regions per retina using a 20 X objective, and automatically quantified with the Fiji plugin ‘Simple RGC’ (Cross et al., 2021 (link)). The representative images shown were acquired on a confocal microscope (Leica Microsystems) using a 40 X objective.
4
Analyzing Retinal Cell Populations and Dendrimer Uptake
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PF-PBS post-fixed retina, lamina and ON frozen sections were analyzed for fluorescent dendrimer signal (D-Cy5), as well as for RGCs (goat anti human Brn3a; Santa Cruz Sc-31984; 1:500 concentration), total inflammatory cells (mouse anti-human IBA1; Millipore MABN92; 1:500 concentration), and astrocytes (glial acidic fibrillary protein [rabbit anti-pig GFAP; Sigma-Aldrich clone GA5; 1:1000 concentration] and aldolase dehydrogenase 1L1 [rabbit anti-mouse Aldh1L1; Abcam ab87117]; 1:1000 concentration). We performed antigen recovery using citrate buffer. Primary antibodies made in different species (mouse antihuman IBA1 and rabbit antimouse Aldh1L1) enabled us to colocalize both Aldh1L1 and IBA1 without difficulty, using fluorescently labeled donkey anti-rabbit (Cy2) and donkey anti-mouse (Cy3) secondary antibodies. Histological analysis was performed using a 4-channel confocal microscope (Olympus E900).
5
Retinal Flat Mount Analysis of Brn3a+ Cells
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Seven days after the intravitreal injections, mice (n = 6 mice/eyes per treatment group) were euthanized, their eyes were enucleated and immediately fixed overnight in 4% paraformaldehyde at 4°C. After three washes with 1x PBS, retinas were carefully separated from the globe and incubated overnight in blocking buffer (5% normal donkey serum and 5% BSA in PBS) at 4°C, cuts were made in the four quadrants (superior, inferior, nasal and temporal) and retinal flat mounts were prepared. The retinal flat mounts were then incubated with the primary antibody, goat anti-Brn3a (1:200, SC-31984, Santa Cruz Biotechnology, Inc.) for three days at 4°C. After three washes with PBS, the flat mounts were incubated overnight in the corresponding secondary antibodies: Alexa 488 conjugated donkey antigoat antibody (1:1000 dilution, A11055, Invitrogen) at 4°C. After washes, the flat mounts were mounted using Prolong Gold anti-fade (Life Technologies) and images were taken using the Keyence fluorescence microscope (Keyence, Osaka, Japan).
6
Retinal Whole-Mount Immunostaining for Brn3a
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Flattened retinal whole-mounts were immersed in 30% sucrose in PBS for 24 h at 4 °C. The retinas were treated with blocking solution (PBS containing 3% donkey serum, 1% bovine serum albumin, 1% fish gel and 0.1% Triton X-100) for 1 h at room temperature. The retinas were incubated with goat polyclonal Brn3a antibody (1:500; sc-31984, Santa Cruz Biotechnology, Dallas, TX, USA) for 3 days at 4 °C. After washing with PBS for 10 min by 3 times, the retinas were incubated with the secondary antibodies, Alexa Fluor-568 donkey anti-goat IgG antibody (1:100; A-11057, Invitrogen, Carlsbad, CA, USA) for 24 h, and subsequently washed with PBS.
7
Retinal Ganglion Cell Visualization
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To visualize RGCs, an immunofluorescent staining for Brn3a was performed on whole-mounted retinas fixed in 4% PFA for 1 hour. In brief, whole-mounted retinas were incubated overnight with goat anti-Brn3a (1:500, Sc-31984, Santa Cruz) in PBS with 2% pre-immune serum and 2% Triton X-100, followed by incubation with an Alexa Fluor-conjugated secondary antibody (Invitrogen). Mosaic pictures of entire retinal whole-mounts were made using a laser confocal scanning microscope (Olympus FV1000), and a maximum intensity projection image of a z-stack of the entire RGC layer (30-40 planes, 3 μm interval) was generated (Fiji software (Schindelin et al., 2012) ). Brn3a-positive RGCs, expressed as number of Brn3a-positive cells per mm 2 , were semi-automatically quantified and isodensity maps were generated, using validated Fiji algorithms, all as described (Geeraerts et al., 2016) (link).
8
Immunostaining of Brn3a and GFAP in Tissue Sections
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For Brn3a immunostaining, sections were incubated with HistoVT One (Nacalai Tesque, Inc., Kyoto, Japan) at 70 °C for 20 min and then with 10% donkey serum and 1% BSA mixture in PBS containing 0.1% Triton X-100 at room temperature (RT) for 2 h. Goat anti-Brn3a antibody (1:400; sc-31984, Santa Cruz Biotechnology, TX) was applied at RT over a two-night period. After three washes in PBS that contained 0.05% Triton X-100, the Alexa Fluor 568 donkey anti-goat IgG conjugate was applied at RT for 1 h.
For glial fibrillary acidic protein (GFAP) immunostaining, sections were incubated with 10% skim milk in PBS at RT for 2 h and then with rabbit anti-GFAP antibodies (1:500; Z0334, Dako, Glostrup, Denmark) overnight at 4 °C. After three washes in PBS, biotinylated goat anti-rabbit IgG antibody (1:200; BA-1000, Vector Laboratories Inc., Burlingame, CA) was applied for 1 h and followed by Cy3 Streptavidin (1:2000; 016–160–084, Jackson ImmunoResearch Laboratories, Inc., PA) for 1 h.
All sections were mounted in the Vectashield Antifade Mounting Medium with DAPI (Vector Laboratories Inc.). Images were obtained by BX60 (Olympus Corporation). Three sections from each eye were observed.
For glial fibrillary acidic protein (GFAP) immunostaining, sections were incubated with 10% skim milk in PBS at RT for 2 h and then with rabbit anti-GFAP antibodies (1:500; Z0334, Dako, Glostrup, Denmark) overnight at 4 °C. After three washes in PBS, biotinylated goat anti-rabbit IgG antibody (1:200; BA-1000, Vector Laboratories Inc., Burlingame, CA) was applied for 1 h and followed by Cy3 Streptavidin (1:2000; 016–160–084, Jackson ImmunoResearch Laboratories, Inc., PA) for 1 h.
All sections were mounted in the Vectashield Antifade Mounting Medium with DAPI (Vector Laboratories Inc.). Images were obtained by BX60 (Olympus Corporation). Three sections from each eye were observed.
9
Comprehensive Antibody Panel for Neural Tissue Analysis
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The following antibodies were used for immunopanning, immunocytochemistry, immunohistochemistry and Western Blot experiments: goat anti-OLIG2 (AF2418, R and D Systems, 1:50), mouse anti-APC (clone CC1, OP80, 1:300, Millipore Sigma), mouse anti-NOGO-A (clone 11C7, gift from M.E. Schwab, 1:3,000), rat anti-MBP (ab7349, Abcam, 1:500), mouse anti-MOG (clone 8–18 C5, 1:1,000, Millipore Sigma), rat anti-GFAP (clone 2.2B10, 13–0300, Invitrogen, 1:1,000), rabbit anti-AQP4 (AB3594, 1:500, Millipore Sigma), rabbit anti-KIR4.1 (APC035, Alomone Labs, 1:3,000), rabbit anti-KIR4.1 (APC-165, Alomone Labs, 1:1,000), rabbit anti-KIR5.1 (APC123, Alomone Labs, 1:500), mouse anti-Neurofilament H (NF-H), nonphosphorylated (clone SMI32, 801701, Biolegend, 1:10,000), mouse anti-Neurofilament H (NF-H), phosphorylated (clone SMI312, 837904, Biolegend, 1:1,000), rabbit anti-IBA1 (019–19741, Wako, 1:500), goat anti-BRN3a (sc-31984, Santa Cruz, 1:200), rabbit anti-KCNQ2 (ab22897, Abcam, 1:200) rabbit anti-CASPR (ab34151, Abcam, 1:1,000), mouse anti-BRDU (347580, BD Biosciences, 1:200), rabbit anti-phospho-Histone H3 (pH3, 9701, Cell Signaling, 1:500), rat anti-CD140a (558774, BD Biosciences, 1:500), mouse anti-β-ACTIN (A5316, Sigma, 1:7,000).
10
Retinal Ganglion Cell Quantification
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Mice were euthanized (pentobarbital, 60 mg/kg body weight, Nembutal, Vetoquinol) and transcardially perfused with saline and 4% phosphate buffered paraformaldehyde (4% PFA, pH 7.4). The eyes were dissected and post-fixed in 4% PFA at room temperature for 1 h. Thereafter, the retina was dissected and post-fixed in 4% PFA at room temperature for another hour. For immunostainings, whole mount retinas were incubated overnight with the primary antibody (goat anti-Brn3a; 1:500, sc-31984, Santa Cruz, diluted in 2% pre-immune serum and 2% triton X-100 (VWR) in PBS) at room temperature. Then, the samples were incubated with an Alexa Fluor® conjugated secondary antibody for 2 h. Mosaic pictures of the entire retinal whole mounts were made using a laser scanning confocal microscope (Olympus FV 1000), and the maximum intensity projection images of z-stacks (step size of 3 µm) were analyzed. The total number of Brn3a+ RGCs was semi-automatically counted in complete retinal flat mounts using a validated Fiji algorithm and expressed as Brn3a+ cell number per mm2, all as previously described [32 (link)].
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