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

نویسندگان

  • Byung Joo Kim
  • So Yeon Lee
  • Hyung Woo Kim
  • Eun-Jung Park
  • Jun Kim
  • Sang Jeong Kim
  • Insuk So
  • Ju-Hong Jeon
چکیده

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.

برای دانلود رایگان متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

منابع مشابه

Differential expression of calbindin D28k, calretinin and parvalbumin in the cerebellum of pups of ethanol-treated female rats.

Three calcium-binding proteins (CaBPs), calbindin D28k, calretinin and parvalbumin, were immunohistochemically examined in the cerebellum of ten-day-old rat pups of ethanol-treated dams. Dams were treated with ethanol during pregnancy and/or lactation. In the cerebellar cortex of the pups from control groups, Purkinje cells with their processes and Golgi cells were positive for calbindin D28k, ...

متن کامل

Calbindin D28k expression and the absence of apoptosis in the cerebellum of Solanum bonariense L-intoxicated bovines.

Solanum bonariense intoxication is characterized by cerebellar neuronal vacuolation, degeneration, and necrosis. Cerebellar Purkinje cells seem especially susceptible, but more research is needed to determine the pathogenesis of neuronal necrosis and the mechanism of Purkinje cell susceptibility. Calbindin D28k (CbD28k) is highly expressed in Purkinje cells and has been used as a marker for nor...

متن کامل

Single course of antenatal betamethasone produces delayed changes in morphology and calbindin-D28k expression in a rat's cerebellar Purkinje cells.

In the current study, we analyzed the impact of antenatal betamethasone on macroscopic cerebellar development, Purkinje cell morphology and the expression of the neuroprotective protein calbindin-D28k. Pregnant rats (Sprague-Dawley) were randomly divided into two experimental groups: control (CONT) and betamethasone-treated (BET). At gestational day 20 (G20), BET dams were subcutaneously inject...

متن کامل

Ataxia and altered dendritic calcium signaling in mice carrying a targeted null mutation of the calbindin D28k gene (calcium-binding proteinymotor coordinationysynaptically evoked calcium transientsycerebellar Purkinje neuronsycalcium imaging)

Intracellular calcium-binding proteins are abundantly expressed in many neuronal populations. Previous evidence suggests that calcium-binding proteins can modulate various neuronal properties, presumably by their action as calcium buffers. The importance of calcium-binding proteins for nervous system function in an intact integrated system is, however, less clear. To investigate the physiologic...

متن کامل

Immunohistochemical localization of calbindin-D28k in the kidney and cerebellum of the porcupines (Hystrix cristata)

The localization of calbindin in the kidney and cerebellum of Hystrix cristata was investigated immunohistochemically using an antiserum against the 28k Da calbindin of chicken duodenum. Calbindin-D28k is an intracellular protein with a high affinity for calcium. This protein is exclusively localized in the distal convoluted tubules of the kidney and in Purkinje cells of the cerebellum. Functio...

متن کامل

ذخیره در منابع من


  با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید

عنوان ژورنال:
  • The Korean journal of physiology & pharmacology : official journal of the Korean Physiological Society and the Korean Society of Pharmacology

دوره 13 5  شماره 

صفحات  -

تاریخ انتشار 2009