M*******C
发帖数: 183 | 1 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
发帖数: 150 | 2 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]. |
M*******C
发帖数: 183 | 3 那看来只能10x看了，如果效率高还好，很多绿细胞，效率不高的话，挺困难，零星的
绿色，也不确定是不是杂质，有的杂质太像细胞了
.
proteins
,
antibody.
www.
【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
发帖数: 150 | 4 find a better microscope that allows you to exam the cells with a higher
magnification? |
g*********e
发帖数: 150 | 5 flow cytometry may be another option |
l***y
发帖数: 638 | 6 悬浮细胞啊？只能facs
Negative
【在 M*******C 的大作中提到】
: 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
|
c******o
发帖数: 1184 | 7 alex 488? It is very stable. If so, go ahead to do the PFA.
Negative
【在 M*******C 的大作中提到】
: 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
|
M*******C
发帖数: 183 | 8 贴壁的
【在 l***y 的大作中提到】
: 悬浮细胞啊？只能facs
:
: Negative
|
M*******C
发帖数: 183 | 9 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
|
