Open AccessOptimized Protocol of Methanol Treatment for Immunofluorescent Staining in Fixed Brain Slices
Author(s) -
Feng Yuan,
Guoxiang Xiong,
Noam A. Cohen,
Akiva S. Cohen
Publication year - 2017
Publication title -
applied immunohistochemistry and molecular morphology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.868
H-Index - 60
eISSN - 1541-2016
pISSN - 1533-4058
DOI - 10.1097/pai.0000000000000293
Subject(s) - paraformaldehyde , immunostaining , methanol , staining , brain tissue , chemistry , biomedical engineering , pathology , anatomy , biology , immunohistochemistry , medicine , organic chemistry
We optimized methanol treatment in paraformaldehyde-fixed slices for immunofluorescent staining of ependymal basal bodies in brain ventricles. As 100% methanol induced severe deformations to the slices (including rolling and folding over), we tried to decrease methanol concentration. We found that 33.3% to 75% methanol could result in ideal immunostaining of basal bodies without inducing obvious deformations. Instead of treating slices at -20°C (without proper cryoprotection measurements) as suggested in previous studies, we carried out methanol treatment at room temperature. Our modified protocol can not only raise immunostaining efficiency in tissue slices, it may also prevent potential freezing damages to the samples.