(No signal was observed through any phase of validation - the "junky" cells lighting up were observed in control tissue 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 sodium citrate buffer, pH 6.0. 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 1:250 – 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 1:2000 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.
Thank you for taking part in our AbTrial program and your valuable feedback about the use of ab10062 in IHC-FrFl. As the antibody had not yet been tested in this application we were unsure what the result might be and we appreciate the time and effort that the reviewer has put in to testing this product in IHC-FrFl. We encourage all researchers to contact us should they wish to test any of our products in untested species or applications, as these may qualify for our AbTrial program.
Dr. Daphne Gill
Submitted Sep 06 2018
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