佐剂性关节炎大鼠脾脏组织GRK2免疫荧光法建立

Establishment of GRK2 immunofluorescence methods in the spleen of rats with adjuvant arthritis

建立了适合佐剂性关节炎大鼠脾脏组织G蛋白偶联受体激酶2(GRK2)免疫荧光测定法。考察了免疫荧光测定的4个关键条件:(1)渗透剂Triton X-100的影响;(2)不同抗原修复液:柠檬酸缓冲液和p H 9.0乙二胺四乙酸(EDTA)缓冲液的影响;(3)5%胎牛血清封闭不同时间(20、30、40、50min)的影响;(4)不同一抗浓度(1∶50、1∶100、1∶200、1∶400、1∶500)的影响。实验结果显示,Triton X-100通透增强了抗原的表达;1∶100的一抗浓度对荧光信号强度最合适;封闭50 min能显著去除背景非特异性染色;柠檬酸缓冲液优于p H 9.0 EDTA缓冲液,提高了抗原表达。

Immunofluorescence assay for G protein-coupled receptor kinase 2 （GRK2） in spleen tissue of rats with adjuvant arthritis was established. Four key conditions of immunofluoreseence assay were investigated： （1） Effect of penetrating agent Triton X-100 on the result; （2） Effects of different antigen repair fluids： Effects of citric acid buff- er and pH 9.0 EDTA buffer; .（3） Effects of different closed times ： 20 rain, 30 rain, 40 rain and 50 rain ; （4） Effects of different primary antibody concentrations： 1 ： 50, 1 ： 100, 1 ： 200, 1 ： 400 and 1 ： 500 . Experimental results showed that Triton X-100 penetration enhancing antigen expression; Citric acid buffer is better than pH 9. 0 EDTA buffer, which improves the expression of antigen; the concentration of 1 ： 100 of primary antibody was most suitable for the intensity of fluorescence signal; 50 min of blocking time could significantly remove background nonspecific staining