Immunohistochemistry free floating abreview for Anti-SOX10 antibody [SOX10/991] - BSA and Azide free

Immunohistochemistry free floating

Rat Tissue sections (Brain)

Brain

24 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: 5% donkey serum, 0.5% BSA, 0.1% PBS-Trit

1/500

1/20000

Abcam antibody:Donkey Anti-Mouse IgG H&L (Alexa Fluor® 488)

(no signal was observed - fluorescing in image is background staining, present in the negative control as well.) Brains cleared with phosphate buffered saline and perfusion fixed with 4% paraformaldehyde were cryoprotected/post-fixed using a 30% sucrose in 10% neutral buffered formalin solution. Brains were frozen in cryomatrix and stored at -80°C before sectioning on a cryostat to 30 µm. Once sectioned, tissue was stored in Millonigs Working Solution until further processed. Sections were washed three times in PBS for 10 minutes each, then epitope retrieval (HIER) was performed on free floating sections at 80°C for 30 minutes in various epitope retrieval buffers (sodium citrate, pH 6.0 sodium citrate buffer, pH 8.0 1 mM EDTA, pH 8.0 10 mM Tris, 1 mM EDTA, 0.05% Tween20, pH 9.0 0.1 M boric acid 1% SDS in PBS and no HIER at all). Tissue was allowed to cool for 20 minutes, then washed with 0.1% PBS-Tween for ten minutes, and blocked (5% donkey serum, 0.5% bovine serum albumin, 0.1% PBS-Triton X100) for two hours at room temperature. Brain sections were incubated overnight in 1:500 – 1:2000 dilution of the primary antibody in blocking buffer at 4°C. The following day, sections were washed with 0.1% PBS-Tween, and twice with PBS for 10 minutes each, followed by incubation with 1:10,000 – 1:20,000 AlexaFluor donkey anti-mouse 488 (ab150105) in blocking buffer for 2 hours. Tissue was then washed with 0.1% PBS-Tween and twice with PBS for 10 minutes before mounting and coverslipping. No signal was observed – small “junky” structures fluoresced but were also unfortunately present in their respective negative controls.

Thank you for your valuable feedback. IHC-free floating was not a tested application for ab212843 and it seems that this antibody does not recognize rat SOX10 under these experimental conditions. Thank you for your time. We hope that this information will be helpful to other researchers.