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Optimized Protocol of Methanol Treatment for Immunofluorescent Staining in Fixed Brain Slices

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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Applied Immunohistochemistry & Molecular Morphology Wolters Kluwer Health

Optimized Protocol of Methanol Treatment for Immunofluorescent Staining in Fixed Brain Slices

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Publisher
Wolters Kluwer Health
Copyright
Copyright © 2016 The Author(s). Published by Wolters Kluwer Health, Inc.
ISSN
1062-3345
eISSN
1533-4058
DOI
10.1097/PAI.0000000000000293
pmid
26509907
Publisher site
See Article on Publisher Site

Abstract

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.

Journal

Applied Immunohistochemistry & Molecular MorphologyWolters Kluwer Health

Published: Mar 1, 2017

References