Optimized Immunohistochemical Analysis of Cerebellar Purkinje Cells Using a Specific Biomarker, Calbindin D28k
The Korean Journal of Physiology and Pharmacology
;
: 373-378, 2009.
Article
Dans Anglais
| WPRIM
| ID: wpr-727509
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.
Texte intégral:
Disponible
Indice:
WPRIM (Pacifique occidental)
Sujet Principal:
Cellules de Purkinje
/
Protéine G liant le calcium S100
/
Cervelet
/
Sensibilité et spécificité
/
Ataxies spinocérébelleuses
/
Filtration
/
Horseradish peroxidase
/
Anticorps
Type d'étude:
Etude diagnostique
/
Guide de pratique
langue:
Anglais
Texte intégral:
The Korean Journal of Physiology and Pharmacology
Année:
2009
Type:
Article
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