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。
【英文摘要】
Immunofluorescence assay for G protein-coupled receptor kinase 2( GRK2) in spleen tissue of rats with adjuvant arthritis was established. Four key conditions of immunofluorescence 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 buffer and p H 9. 0 EDTA buffer;(3) Effects of different closed times: 20 min,30 min,40 min and 50 min;(4) Effects of different primary antibody concentrations: 1 ∶ 50,1 ∶ 100,1 ...