带荧光的siRNA转染后，固定会影响荧光检测否？# Biology - 生物学
v*n
1 楼
因为工作的原因，要去欧洲（较长时间）。在BOA有一点存款，所以除了身上带一些换
的欧元之外，有什么比较保值，比较安全的方法？
转帐？还是别的什么方法？
的欧元之外，有什么比较保值，比较安全的方法？
转帐？还是别的什么方法？
M*C
2 楼
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
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].
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
4 楼
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,
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
5 楼
find a better microscope that allows you to exam the cells with a higher
magnification?
magnification?
g*e
6 楼
flow cytometry may be another option
l*y
7 楼
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
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
8 楼
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
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
9 楼
贴壁的
【在 l***y 的大作中提到】
: 悬浮细胞啊？只能facs
:
: Negative
【在 l***y 的大作中提到】
: 悬浮细胞啊？只能facs
:
: Negative
M*C
10 楼
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
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
相关阅读
Re: 大家有没有这样的情况？down with cheaters Re: 急：bioinformatic的请进Re: 大虾救命Re: 请问这里有学药理学的前辈吗?(药理学如何帮助我们理解脑?)Re: 想知道细胞转染的具体机制和过程JCR: Chemistry影响因子Re: 弱弱的问题：能检测出来吗？Re: helpScientists who pioneered arthritis drug强烈推荐Clustal WRe: help in multiple正确答案Re: ===这里有没有高手呀?Salary Survey Shows Who’s in the Genome[转载] SARS is sequencedwestern战斗的个人心得兼谢各位指点the way to do itRe: PCR产物酶切一问Re: PCR产物纯化方法Nature signaling gatewayRe: 硫氰酸胍－酚法抽提植物RNARe: pCMV5 vector map,help plsBreaking news