Optimized Immunohistochemical Analysis of Cerebellar Purkinje Cells Using a Specific Biomarker, Calbindin D28k.
10.4196/kjpp.2009.13.5.373
- Author:
Byung Joo KIM
1
;
So Yeon LEE
;
Hyung Woo KIM
;
Eun Jung PARK
;
Jun KIM
;
Sang Jeong KIM
;
Insuk SO
;
Ju Hong JEON
Author Information
1. School of Korean Medicine, Pusan National University, Yangsan 626-870, Korea.
- Publication Type:Original Article
- Keywords:
Purkinje cell;
Cerebellum;
Calbindin D28K;
Immunohistochemical analysis
- MeSH:
Antibodies;
Calcium-Binding Protein, Vitamin D-Dependent;
Cerebellum;
Filtration;
Horseradish Peroxidase;
Purkinje Cells;
Sensitivity and Specificity;
Spinocerebellar Ataxias
- From:The Korean Journal of Physiology and Pharmacology
2009;13(5):373-378
- CountryRepublic of Korea
- Language:English
-
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 micrometer 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 disorders and to evaluate the efficacy of therapeutic strategies for treating these diseases.
