【资源】冰冻切片DAB染色步骤，屡试不爽！

DAB Staining
1.  Cut 40μm cryostat free floating sections.
2.  Rinse sections with 1% H2O2 in TBS for 30 min at RT.
3.  Rinse sections with TBS, 5 min ×3.
4.  Incubate sections in 0.5% Triton-x-100 in TBS for 60 min at RT.
5.  Incubate sections with(10% donkey serum +0.5% Triton-x-100)(block solution) in
TBS for 90 min at RT(pH=7.2).
6.  Aspirate Block solution. Incubate sections with
Mouse monoclonal anti-CR(Chemicon, catalog No.MAB1568,lgG1),1/2,000)
Or
Goat polyclonal anti-DCX(Santa Cruz, catalog No. sc-8066;1/200)
Diluted in block solution and overnight at 4℃.
7.  Balaced at RT for about 30 min*1.
8.  Rinse with TBS,10 min×4.
9.  Incubate sections with second antibodies:
For visualizing CR, use DAM-Biotin(Jackson ImmunoResearch,
Catalog No.715-065-151,IgG(H+L),由于本实验室稀释二抗时添加了等体积的甘油，所以在使用本实验室的二抗时稀释倍数为1/500);
For visualizing DCX, use DAM-Biotin(Jackson ImmunoResearch,
Catalog No.715-065-147, IgG(H+L),1/1,000, 由于本实验室稀释二抗时添加了等体积的甘油，所以在使用本实验室的二抗时稀释倍数为1/500).
Diluted in(5% donkey serum+0.5% Trixton-x-100)。
Incubate RT for 2 hours.
10.  Rinse with TBS,10 min×4.
11.  Incubate sections with third antibodies: Peroxidase-conjugated Streptavidin(HRP)
(Jackson ImmunoResearch, Catalog No.016-030-084,1/250(2μg/ml)
Diluted in TBS and incubate for 1 hour at RT.
12.  Rinse with TBS,10 min×3.
13.  DAB staining:使用中杉试剂盒，1滴/1.5ml，37℃显色5-7分钟左右（注意避
光），呈色后在TBS中终止反应（DAB有毒，所以操作时戴手套，操作结束
后彻底洗手）。