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.