消除小鼠肝脏冰冻切片自发荧光4种封闭方法的比较

目的 通过对小鼠肝脏冰冻切片进行不同的封闭处理，探寻消除小鼠肝脏自发荧光的最佳方法，以降低对免疫荧光阳性信号的干扰，提高免疫荧光结果的准确性。方法 （1）经脾静脉注射肝脏荷瘤裸小鼠：将Hepa1-6-GFP细胞通过脾脏注射方法使其在雄性无胸腺BALB/c裸小鼠肝脏定植，并将肝脏进行冰冻连续切片，对切片分别进行AB液（内源性生物素封闭液）、blocking、AB液+blocking、丙酮+AB液+blocking封闭处理，然后检测小鼠肝脏冰冻切片自发荧光。（2）C57BL/6小鼠：对C57BL/6小鼠肝脏进行冰冻连续切片，对切片分别进行上述4种封闭处理，用F4/80标记肝巨噬细胞（Kupffer细胞），然后检测小鼠肝脏冰冻切片自发荧光。结果 肝脏组织冰冻切片免疫荧光染色时，4种封闭处理都可以减弱肝脏切片的自发荧光，但丙酮+AB液+blocking封闭处理效果最佳。结论 丙酮+AB液+blocking联合应用对小鼠肝脏组织冰冻切片自发荧光的消除效果最好。

Comparison of four blocking methods for eliminating autofluorescence of mouse liver frozen sections

Objective To compare different blocking methods for eliminating autofluorescence of mouse liver frozen sections, so as to find the best method to reduce the interference to immunofluorescence positive signals and improve the accuracy of immunofluorescence. Methods Intrasplenic injection-liver colonization nude mice:Hepa1-6-GFP cells were intrasplenically injected into male athymic BALB/c nude mice to create liver colonization models. Liver tissues were frozen and continuously sectioned. Sections were blocked with AB reagent (A reagent:streptavidin reagent, B reagent:biotin reagent), blocking buffer, AB reagent+blocking buffer, or acetone+AB reagent+blocking buffer, and then the autofluorescence of the frozen sections was detected. C57BL/6 mice:the liver tissues of C57BL/6 mice were frozen and continuously sectioned, and then the sections were blocked with AB reagent, blocking buffer, AB reagent+blocking buffer, or acetone+AB reagent+ blocking buffer. Liver macrophages (Kupffer cells) were labeled with F4/80, and then the autofluorescence of mouse liver frozen sections was detected. Results In the immunofluorescence staining of liver tissue frozen sections, all the above four blocking methods could reduce the autofluorescence of liver sections, and acetone+AB reagent+blocking buffer group had the best effect. Conclusion The combined buffers (acetone+AB reagent+blocking buffer) has the best effect in eliminating the autofluorescence of mouse liver frozen section.