Author(s): QIN You, CHEN Yongchang
Pages: 385-388
Year: 2006 Issue:  5
Journal: Journal of Zhenjiang Medical College
Keyword:  fixative; immunofluorescence microscopy; TritonX-100; RhoA;
Abstract: Objective: To observe the effect of different fixatives on the location of RhoA protein and find the optimum fixative for the localization of RhoA in SGC7901 cells by immunofluorescence microscopy.Methods: The subcellular location of RhoA protein in cultured SGC7901 cells fixed with different fixatives such as 2% paraformaldehyde,4% formaldehyde,methanol,acetone,and 10% trichloroacetic acid(TCA) was studied by immunofluorescence microscopy. Results: Cells fixed by 2% paraformaldehyde without TritonX-100 treatment showed accumulation of RhoA in nucleus but little distribution in cytoplasm.After penetrating with TritonX-100,RhoA protein could not be detected in cytoplasm and was more accumulated in nucleus.Cells fixed by 4% formaldehyde without TritonX-100 penetrating showed nuclear distribution of RhoA.After penetrating with TritonX-100,the staining became clearer.Fixation of SGC7901 cells with methanol or acetone without TritonX-100 penetrating showed that RhoA distributed mainly in the cytoplasm.After penetrating with TritonX-100,RhoA staining could be seen in nucleus and became weaker in cytoplasm.In addition,in cells fixed by 10% TCA without TritonX-100 penetrating,RhoA was stained blurredly and could be seen in both cytoplasm and nucleus.After TritonX-100 penetrating,the staining became clear and the distribution was reduced in cytoplasm and increased in nucleus. Conclusion: Different fixatives and penetrating with TritonX-100 or not are important for localizing RhoA protein by immunofluorescence microscopy.