I perfused the mice, fixed the brains and used the Microslicer to cut to slides then used the avidin-biotin-peroxidase (ABC method) to do immunohistochemistry analysis.
the method for details I used are from these three articles.(at last of my question)
Then you can see the photo:
In the upper part of the photo I successfully did it before, but now I cannot get the result and I don’t know why. You can see the rest two slides are too dark and may full of nonspecific binding. it sure that these slides cannot be used. Thank you very much if you can help me.
Koike K, et al (2005) The immunophilin ligand FK506 protects against methamphetamine-induced dopaminergic neurotoxicity in mouse striatum. Neuropharmacology 48:391–397.
Zhang L, et al (2006) Protective effects of minocycline on behavioral changes and neurotoxicity in mice after administration of methamphetamine. Prog Neuropsychopharmacol Biol Psychiatry 30:1381–1393.
Ren Q, Zhang JC, Ma M, Fujita Y, Wu J, Hashimoto K (2014) 7,8-Dihydroxyflavone, a TrkB agonist, attenuates behavioral abnormalities and neurotoxicity in mice after administration of methamphetamine. Psychopharmacology (Berl) 231:159–166.
Mohamed Mahdy
Immunohistochemistry of DAT
Free-floating sections were treated with 0.3% H2O2 in 0.05M Tris-HCl saline (TBS) for 30 min and blocked in TBS containing 0.2% Triton X-100 (TBST) and 1.5% normal serum for 1 hour, at room temperature. Samples were then incubated for 36 hours at 4°C, with rat anti-DAT antibody. The sections were washed three in TBST, and processed according to the avidin-biotin-peroxidase method (Vectastain Elite ABC, Vector Laboratories, Inc., Burlingame, CA, USA). Sections were then incubated for 5 minutes in a solution of 0.15 mg/ml diaminobenzidine, containing 0.01% H2O2. The sections were mounted on gelatinized slides, dehydrated, cleared, and coverslipped under Permount® (Fisher Scientific, Fair Lawn, NJ, USA).
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