Research Of Immunofluorescence Staining For Detection Of Transcription Factor In Mouse Oocytes And Early Embryos

The aim of this experiment was to study the effective method of fixation and penetration before immunofluorescence staining in oocytes and early embryos to raise the effect of immunofluorescence staining in them.Simultaneously,the aim was to investigate the role of PolⅡduring the implantation and development of mouse oocytes and early embryos by studying the expression of PolⅡin them.Builds the solid foundation for detection the protein and transcription factor accurately in mouse oocytes and early embryos on a large scale.The materials were mouse oocytes and early embryos which treated by four different methods of fixation and penetration.Method A:Oocytes and early embryos were fixed in 4%paraformaldehyde(PFA)/PBS for 1 hour at room temperature, then permeated in 0.5%Triton X-100 for 10 minutes.Method B:Oocytes and early embryos were permeated in 0.5%Triton X-100 for 10 minutes,then fixed in 4% paraformaldehyde(PFA)/PBS.Method C:Oocytes and early embryos were fixed in 1%PFA while permeated in 0.2%Triton X-100 at the same time for 1 hour.Method D: Oocytes and early embryos were fixed in precooled methanol(-20℃) for 15 min,then permeated in 0.5%Triton X-100 for 10 minutes.Ten transcription factors(TFⅡA,TFⅡB,TAF1,TAF4,PolⅡ,BRF1,Mecp2,MBD2ab,HP1αand HP1β)in mouse oocytes were analyzed by immunofluorescence staining.The results indicated that only by using the method C the fluorescence signal of all target transcription factors were detected and marked,the rest three methods can detected the fluorescence signal of the transcription factor partially.To analyse the most typical TFⅡB with fine fluorescence signal distribution pattern and dynamic change carefully,it's indicated that oocyte's shape was completely and distribution pattern of ten transcription factors antibody signal were fine by using method C,the fluorescence signal was clear and can observe the signal change dynamic with the cell nucleus mature also.Although the nucleolus can be observed in oocytes treated by other three methods,and also the signal can be beobserved in the nuclear area distribution basic evenly,but not clearly displayed the nucleolus fluorescence signal if there were signals in the distribution and dynamic change.Fluorescence staining didn't well.The oocytes and early embryos treated by four methods were observed in light microscope and compared sharp with stages of GV,MⅠ,MⅡ,2-cells,it's showd that oocyte's shape was completely by using method C.In the oocytes of GV stage, the structure of nucleus and nucleolus were clearly and the distribution of the cytoplasm were evenly,also the sharp were plumply.The granular cytoplasm can be seen in the oocytes of MⅠstage and the ZP were firms,clear and uniform thickness. The first polar body in the oocytes of MⅡstage and the polar body in the embryos of 2-cells stage were keeped well.The oocytes and early embryos treated by other three methods were out of sharp and the structure of nucleolus,ZP and polar body in different stages were damaged.Particularly,the greatest damage of sharp was by using method D indicated that mouse oocytes and early embryos fixed in precooled methanol(-20℃) were inapplicable.Analysis the expression of PolⅡin the mouse oocytes and early embryos:the signal were not detected in the oocytes aider GVBD until MⅡstage and also in the embryos of Ana,PN1 stage.But the signal can be detected in the embryos of PN2 and PN3 stages after fertilization.The embryos of PN4,PN5 and 2-cells stages were all positive.This expression was match of the early gene silencing,which supported the hypothesis on the silence of transcription.