Brn3a is accepted as a good marker for RGC17 (link) and was used for RGC analysis in MSC-treated and untreated EAE mice and controls thirty two days after induction. Eyes of 14 mice per group were harvested, fixed in 4% paraformaldehyde for 2 hours, and prepared for wholemount staining. Retinas were permeabilized in 0.3% Triton X-100/PBS for 6 hours followed by a freeze and thaw cycle at −80°C. One percent bovine serum albumin (BSA)/0.3% Triton X-100/PBS was used for 1 hour blocking at room temperature. Retinas were incubated with goat-anti Brn3a primary antibody (Santa Cruz, TX; diluted 1:200 in 1%BSA/0.3% Triton X-100/1% DMSO/PBS) at 4°C for 48 hours on a rocker platform. After washes in PBS, a donkey anti-goat Alexa Fluor 546 secondary antibody solution (Invitrogen, Carlsbad, CA; 1:200 in PBS) was applied for 3 hours at room temperature in the dark. Following PBS washes, retinas were mounted with Vectashield (Vector Laboratories, Burlingame, CA). Twelve images from each retina were taken at predetermined locations peripherally (5/6), mid-peripherally (3/6), and centrally (1/6) at 20x magnification using an Olympus BX41 microscope (Olympus, Center Valley, PA). Brn3a+ cells were counted manually by an observer masked to the protocol using the cell counter plug in ImageJ software (National Institutes of Health, Bethesda, MD). RGC numbers were normalized to density per mm2.
Goat anti brn3a primary antibody
Manufactured by Santa Cruz Biotechnology
Sourced in Australia
Goat-anti Brn3a primary antibody is a polyclonal antibody that binds to the Brn3a transcription factor, also known as POU4F1. This antibody can be used to detect and analyze the expression of Brn3a in various biological samples.
Automatically generated - may contain errors
3 protocols using goat anti brn3a primary antibody
1
Quantifying Retinal Ganglion Cells in EAE
2
Retinal Ganglion Cell Quantification Protocol
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Whole flat-mounted retinas were permeabilized in 0.5% Triton X-100 in PBS (PBST) for 15 min and then blocked in 5% bovine serum albumin (BSA) for 1 h. Then, the retinas were incubated in goat-anti-Brn3a primary antibody (1 : 200 dilution; SC-31985; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) overnight at 4 °C in order to label RGCs. Following extensive washing in PBS, the specimens were incubated with Cy3-labeled donkey anti-goat secondary antibody (Jackson ImmunoResearch, West Grove, PA; 1 : 100) for 1 h at room temperature. Finally, the retinas were flat-mounted with the vitreous side up on a glass slide using an antifading mounting medium and then observed and imaged using a fluorescent digital microscope (BX53, Olympus, Tokyo, Japan). The number of RGCs in each specimen was counted using image analysis software (Image Pro-Plus 6.0; Media Cybernetics, Inc., MD, USA). As described previously [20 (link)], central and midperipheral RGC numbers were obtained in the area at a distance of 1 or 3 mm from the center of the retina specimen, respectively. Four random areas (one in each quadrant; 200× magnification) in the central retina and four random areas (one in each quadrant; 200× magnification) in the midperipheral retina were selected for analysis, and the average RGC counts per mm2 retinal area, known as the RGC density, was calculated.
3
Retinal Wholemount Immunolabeling Protocol
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All rats were terminally anesthetized by transcardial perfusion using physiological saline. Whole eyes were removed and placed in 10% neutral buffered formalin for one hour. Posterior eye-cups were carefully dissected and each retina was prepared as a flattened wholemount via four relaxing incisions. Retinas were permeabilized with phosphate buffered saline (PBS; 137 mM NaCl, 5.4 mM KCl, 1.28 mM NaH2PO4, 7 mM Na2HPO4; and pH 7.4) containing 1% Triton X-100 (PBST-1%), blocked in PBST-1% containing 3% (v/v) normal horse serum, then incubated for three days at 4°C in the same solution containing either goat anti-Brn3a primary antibody (1:600; SC-31984; Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit anti-RBPMS primary antibody (1:500; ABN1362; Merck Millipore, Bayswater, Victoria, Australia).
After multiple washes with PBST, wholemounts were incubated overnight at 4˚C with alexa fluor 488 or 594-conjugated donkey anti-goat secondary antibody (for Brn3a) or alexa fluor 488 or 594-conjugated donkey anti-rabbit secondary antibody (for RBPMS; 1:500; Invitrogen, Mulgrave, Victoria, Australia), before rinsing in PBS and mounting using anti-fade mounting medium.
After multiple washes with PBST, wholemounts were incubated overnight at 4˚C with alexa fluor 488 or 594-conjugated donkey anti-goat secondary antibody (for Brn3a) or alexa fluor 488 or 594-conjugated donkey anti-rabbit secondary antibody (for RBPMS; 1:500; Invitrogen, Mulgrave, Victoria, Australia), before rinsing in PBS and mounting using anti-fade mounting medium.
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