Optimized immunohistochemical analysis of cerebellar purkinje cells using a specific biomarker, calbindin d28k.
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 disorders and to evaluate the efficacy of therapeutic strategies for treating these diseases.
Kim
Byung Joo
BJ
School of Korean Medicine, Pusan National University, Yangsan 626-870, Korea.
Lee
So Yeon
SY
Kim
Hyung Woo
HW
Park
Eun-Jung
EJ
Kim
Jun
J
Kim
Sang Jeong
SJ
So
Insuk
I
Jeon
Ju-Hong
JH