Application
Immunohistochemistry free floating
Sample
Rat Tissue sections (Brain)
Specification
Brain
Other product details
Incubation time
24 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: 5% donkey serum, 0.5% BSA, 0.1% PBS-Trit
Dilution
1/1000
Secondary antibody
Dilution
1/5000
Name
Abcam antibody:Donkey Anti-Rabbit IgG H&L (Alexa Fluor® 594)
Additional data
Additional Notes
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 Tris-EDTA buffer, pH 9.0 (10 mM Tris, 1 mM EDTA, 0.05% Tween20). It should be noted that HIER at 80°C for 45 minutes produced clearer signal but caused some damage to the brain sections. 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 1:1000 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 1:5000 AlexaFluor donkey anti-rabbit 594 (ab150076) 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.
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投稿 Aug 30 2018