Thirty days post induction, animals were euthanized, and eyes and ONs isolated and post-fixed in 4% paraformaldehyde (PF)-PBS (pH 7.4). PF-PBS-fixed retinas were isolated, and RGCs were immunostained using a primary goat polyclonal anti-Brn3a antibody (Santa Cruz Biotechnology, Dallas, TX), followed by fluorescent labeling with a donkey anti-goat Cy3 secondary antibody (Jackson Immunoresearch, West Grove, PA), flat-mounted and stereologically counted as previously described, using a Nikon Eclipse E800 fluorescent microscope (Nikon, Melville NY) with motorized stage, driven by a stereological imaging package (StereoInvestigator, Ver 10.0; Microbrightfield Bioscience, Williston, VT). Stereological analysis was performed using the Stereo Investigator 10 package, with counts in each eye greater than that required by the Schmitz-Hof equation for statistical validity [21 (link)].
Anti brn3a antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The Anti-Brn3a antibody is a primary antibody that specifically recognizes the Brn3a protein. Brn3a is a transcription factor that plays a crucial role in the development and survival of specific neuronal cell types. The antibody can be used to detect and study the expression and localization of Brn3a in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse e800 fluorescent microscope, by Nikon (1 mentions)
Donkey anti goat cy3 secondary antibody, by Jackson ImmunoResearch (1 mentions)
Alexa fluor 568 donkey anti goat igg antibody, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Spinning disc confocal microscope, by Olympus (1 mentions)
Anti actin antibody, by Merck Group (1 mentions)
Anti cleaved caspase 3 antibody, by Cell Signaling Technology (1 mentions)
Prolong gold antifade mounting media, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
4 protocols using anti brn3a antibody
1
Quantitative Analysis of Retinal Ganglion Cells
2
Immunohistochemical Analysis of Retinal Neurons
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Retinas from enucleated eyes were dissected as flattened whole mounts at 2 weeks after ischemia. Retinas were immersed in PBS containing 30% sucrose for 24 h at 4 °C. The retinas were blocked in PBS containing 3% donkey serum, 1% bovine serum albumin, 1% fish gel and 0.1% Triton X-100 for 1 h, and then incubated with goat polyclonal anti-Brn3a antibody (1 : 500; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 3 days at 4 °C. After several wash steps, the retinas were incubated with the secondary antibodies, Alexa Fluor-568 donkey anti-goat IgG antibody (Invitrogen, Carlsbad, CA, USA) for 24 h, and subsequently washed with PBS. The retinas were counterstained with the nucleic acid stain Hoechst 33342 (1 μg/ml; Invitrogen) in PBS. Images were captured with a spinning-disc confocal microscope (Olympus America Inc., Center Valley, PA USA) equipped with a high-precision closed loop XY stage and closed loop Z control with commercial mosaic acquisition software (MicroBrightField; MBF Bioscience Inc., Williston, VT, USA). The microscope is equipped with high-resolution high-sensitive CCD camera for high-speed mosaic acquisition.
3
Antibody Validation for Neuronal Markers
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Anti-CHOP and anti-CHOP antibodies were purchased from Chemicon; anti-actin antibody from Millipore; anti-Brn3a antibody from Santa Cruz; and anti-HPC-1 antibody from Sigma-Aldrich; anti-cleaved caspase-3 antibody was purchased from Cell Signaling Technology.
4
Retinal Immunostaining of Hexb Knockout Mice
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Following enucleation whole eyes from Hexb−/− mice (n = 3) and WT littermate controls (n = 3) were snap frozen on dry ice and stored at −80 °C. Eyes were then post fixed in 4% methanol free paraformaldehyde in PBS for 16 h. Subsequent immunostaining of retina cryostat sections and whole retina flatmounts was performed as described previously [30] (link), [31] . Rabbit polyclonal anti-melanopsin antibody (1:2500, UF006, advanced Targeting Systems) and goat polyclonal anti-Brn3a antibody (1:1000, sc-31985, Santa Cruz Biotech) were incubated overnight (retinal sections) or for 3 days (retina flatmounts) at 4 °C in PBS with 0.2% Triton-X and 2% donkey serum. Donkey anti-rabbit Alexa 568 and donkey anti-goat Alexa 488 secondary antibodies diluted in PBS 0.2% Triton-X and 2% donkey serum (1:200) were incubated for 2 h at 22 °C. For staining of retina flatmounts levels of Triton-X were increased to 1%. All wash steps were performed using PBS with 0.05% Tween-20. Samples were mounted in Prolong Gold anti-fade mounting media containing DAPI (Life Technologies).
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