Optimized Protocol of Methanol Treatment for Immunofluorescent Staining in Fixed Brain Slices.
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.
*Department of Orthopedics, Hospital of Tongji University, Shanghai, China †Division of Neurology, Children's Hospital of Philadelphia ‡Philadelphia Veterans Affairs Medical Center Departments of §Otorhinolaryngology-Head and Neck Surgery ∥Pediatrics, Perelman School of Medicine, University of Pennsylvania; Philadelphia, PA.
Feng
F
Yuan
Guoxiang
G
Xiong
Noam A
NA
Cohen
Akiva S
AS
Cohen
D001921:Brain / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D000432:Methanol / Q000737:chemistry*
D000432:Methanol