Korean J Physiol Pharmacol.  2009 Oct;13(5):373-378. 10.4196/kjpp.2009.13.5.373.

Optimized Immunohistochemical Analysis of Cerebellar Purkinje Cells Using a Specific Biomarker, Calbindin D28k

Affiliations
  • 1School of Korean Medicine, Pusan National University, Yangsan 626-870, Korea.
  • 2Department of Physiology, College of Medicine, Seoul National University, Seoul 110-799, Korea. jhjeon2@snu.ac.kr

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.

Keyword

Purkinje cell; Cerebellum; Calbindin D28K; Immunohistochemical analysis

MeSH Terms

Antibodies
Calcium-Binding Protein, Vitamin D-Dependent
Cerebellum
Filtration
Horseradish Peroxidase
Purkinje Cells
Sensitivity and Specificity
Spinocerebellar Ataxias
Antibodies
Calcium-Binding Protein, Vitamin D-Dependent
Horseradish Peroxidase

Figure

  • Fig. 1. Sensitivity and specificity of IHC methods. The expression of CaB in cerebellar specimens was analyzed by the HRP method without (A) or with (B) 1:500-diluted anti-CaB antibody (Sigma) and stained using unfiltered AEC solution. CaB was captured by two different commercial source antibodies, one from Sigma (C) and another from Chemicon (D), for 1 h at RT. Antibodies were probed by HRP polymer-conjugated secondary antibody (HRP-Pol). M, molecular layer; P, Purkinje cell layer; G, granular cell layer; W, white matter region. Scale bar=100 μm. The data shown are representative results of three to five independent experiments.

  • Fig. 2. Increased sensitivity of IHC analysis by filtrating AEC solution. CaB expression in cerebellar specimens was analyzed by the HRP-Pol method without (A) or with (B) 1:500-diluted anti-CaB antibody (Sigma). AEC solution was filtrated with a 0.22 μm syringe filter before adding to tissue specimens. Scale bar=100 μm. The data shown are representative results of three to five independent experiments.

  • Fig. 3. Determination of the optimal titer of primary antibody. Serially diluted primary antibodies to CaB were used to determine the optimum titer (B∼E). The HRP-Pol method with filtrated dye was employed for IHC analysis. Staining specificity was verified by omitting the incubation step with primary antibody (A). Cerebellar specimens were incubated with primary antibody for 1 h at RT (A∼E) or for 16 h at 4°C (F). Scale bar=100 μm. The data shown are representative results of three to five independent experiments.

  • Fig. 4. Determination of the optimal titer of HRP polymer-conjugated secondary antibody. Serially diluted HRP polymer-conjugated secondary antibodies were used to determine the optimum titer (A∼F). The cerebellar specimens were incubated with primary antibody to CaB for 1 h at RT. Scale bar=100 μm. The experimental data shown are representative results of three to five independent experiments.


Reference

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