Optimized immunohistochemical analysis of cerebellar purkinje cells using a specific biomarker, calbindin d28k.

The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology
Byung Joo KimJu-Hong Jeon

Abstract

Cerebellar Purkinje cells (PCs) play a crucial role in motor functions and their progressive degeneration is closely associated with spinocerebellar ataxias. Although immunohistochemical (IHC) analysis can provide a valuable tool for understanding the pathophysiology of PC disorders, the method validation of IHC analysis with cerebellar tissue specimens is unclear. Here we present an optimized and validated IHC method using antibodies to calbindin D28k, a specific PC marker in the cerebellum. To achieve the desired sensitivity, specificity, and reproducibility, we modified IHC analysis procedures for cerebellar tissues. We found that the sensitivity of staining varies depending on the commercial source of primary antibody. In addition, we showed that a biotin-free signal amplification method using a horseradish peroxidase polymer-conjugated secondary antibody increases both the sensitivity and specificity of ICH analysis. Furthermore, we demonstrated that dye filtration using a 0.22 microm filter eliminates or minimizes nonspecific staining while preserving the analytical sensitivity. These results suggest that our protocol can be adapted for future investigations aiming to understand the pathophysiology of cerebellar PC disord...Continue Reading

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Citations

Oct 25, 2014·Acta Neuropathologica·Manja SchubertChristian A Vedeler
Nov 8, 2012·Chronobiology International·Martin F RathMorten Møller
Jun 23, 2016·Journal of Molecular Psychiatry·Stephen Sai FolmsbeeCara J Gottardi
May 6, 2019·International Journal of Molecular Sciences·Richard FairlessRicarda Diem
Apr 22, 2021·International Journal of Toxicology·Anshul SrivastavaJoseph P Hanig

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