为了检测转的效率,用带绿荧光488的CTRLsi (Qiagen 1027284, All Stars Negative CTRLsi AF 488) 转染细胞,两天后看绿荧光,如果活细胞在PBS里,导致只能看10x物 镜,否则会接触液面,并且由于没有DAPI染核,有时难以判断看到的绿色是杂质还是细 胞. 但不懂,如果PFA固定,染DAPI,会不会影响对绿荧光的检测。问了周围两个人, 一个说不要固定,另一个说固定没问题。thx
g*e
3 楼
Formaldehyde, glutaraldehyde, alcohol, etc. does not "quench" fluorescence. Instead, it denatures the GFP such that it no longer functions and fluoresces. Any remaining fluorescence is either non-specific (many proteins auto-fluoresce after fixation) or it is a few GFP molecules that retain their original conformation. If you fix the tissue, then as Ann Cantereau states, you MUST use an anti-GFP antibody to detect the GFP. At that point, you are "seeing" the GFP by whatever tag you have on the secondary antibody.
GFP fluorescence after fixation - ResearchGate. Available from: https://www. researchgate.net/post/GFP_fluorescence_after_fixation10 [accessed Mar 28, 2017].
【在 g*********e 的大作中提到】 : Formaldehyde, glutaraldehyde, alcohol, etc. does not "quench" fluorescence. : Instead, it denatures the GFP such that it no longer functions and : fluoresces. Any remaining fluorescence is either non-specific (many proteins : auto-fluoresce after fixation) or it is a few GFP molecules that retain : their original conformation. If you fix the tissue, then as Ann Cantereau : states, you MUST use an anti-GFP antibody to detect the GFP. At that point, : you are "seeing" the GFP by whatever tag you have on the secondary antibody. : : GFP fluorescence after fixation - ResearchGate. Available from: https://www. : researchgate.net/post/GFP_fluorescence_after_fixation10 [accessed Mar 28,
g*e
5 楼
find a better microscope that allows you to exam the cells with a higher magnification?
对,AlexFluor 488. 问了Qiagen的技术服务,回信说得固定,那就简单了。 Yes. It is hard to visualize with PBS. It is also difficult to see the green fluorescence without washing with PBS due to excess non-specific fluorescence. Yes. It is best to wash with PBS and fix to visualize the green fluorescence.
【在 c******o 的大作中提到】 : alex 488? It is very stable. If so, go ahead to do the PFA. : : Negative