求助IHC或者IF fixation的方法 - 未名空间MITBBS历史存档

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求助IHC或者IF fixation的方法

求助IHC或者IF fixation的方法# Biology - 生物学

l*i2011-04-18 07:04

1 楼

如果爸爸一签过后，妈妈去中信续签，那妈妈的邀请信需要说明为什么之前说爸爸一个
去，后来变成两人都要来吗？
因为老爸一签，老妈是续签，我独生子女父母经济也很一般，觉得先让爸爸去签说是一
个人来探亲比较保险。爸爸签过了妈妈再去中信签，不知道这样靠谱不？在妈妈的邀请
信里面要不要说明原因，为什么之前爸爸面签的时候说一个人探亲，后来妈妈也要来？
请给点意见！！！谢谢

s*92011-04-18 07:04

2 楼

IHC stain一个ER上膜蛋白，自己做的多克隆抗体（rabbit），western效果很好，size
和报道的一直，KO tissue没有带。但是IHC一直不成功，IHC用的是frozen tissue，
acetone fixation，fast blue development。
想请教:
１。这种ER上的膜蛋白用什么方法fix比较好。我用cold acetone 5min，也试过一次4
％PFA。似乎效果都不好，和KO没有区别。
２。有什么方法可以提高信号强度。有成熟的tyramide protocol可以提供吗?

h*o2011-04-18 07:04

3 楼

Are you sure it is the fixation problem?
Have you tried with antigen retrieval.

s*92011-04-18 07:04

4 楼

不清楚问题出在哪里？
没事试antigen retrieval.以为只有石蜡的才需要antigen retrivel,冰冻的片子不需
要。

【在 h*******o 的大作中提到】

: Are you sure it is the fixation problem?
: Have you tried with antigen retrieval.

g*02011-04-18 07:04

5 楼

an antibody good for western-blotting is not necessary a good one for IHC or
IF. You can try it on cell culture first. How about IP with KO as control?
It is definitely a way to go try different antigen retrieval methods.
It is also useful to try different blocking reagents.

【在 s****9 的大作中提到】

: 不清楚问题出在哪里？
: 没事试antigen retrieval.以为只有石蜡的才需要antigen retrivel,冰冻的片子不需
: 要。

s*92011-04-18 07:04

6 楼

I understand it can be the problem of the antibody and will never be solved,
but want to try as hard as possible.
Could you suggest a few antigen retrieval method? Do you think 5min acetone
fixation is enough to expose the antigen epitope?
Very few background on the staining. Do you think it is worthy to try
different blocking reagents?
Thanks so much. Really appreciate your suggestions.

or

【在 g****0 的大作中提到】

: an antibody good for western-blotting is not necessary a good one for IHC or
: IF. You can try it on cell culture first. How about IP with KO as control?
: It is definitely a way to go try different antigen retrieval methods.
: It is also useful to try different blocking reagents.

c*z2011-04-18 07:04

7 楼

以前做过一个膜蛋白
IHC就是和其他蛋白一样的方法
IF只用甲醇(fix+通透)
做出来都是golgi pattern 没有在细胞膜看到任何东西

g*02011-04-18 07:04

8 楼

I had an antibody which could only recognize the PFA fixed sample but not
the one fixed by cold methanol. You may try the trick, but the point is
there may not be "logic"enoughto figure out experimental problems, so don't
limit yourself to any possibility if you really want to push it.
Further, I think you should do IP or westernblotting on native gel to see if
what the antibody recognize. Meanwhile, you can try stain the protein
overexpressed in cell culture.
I know a good antibody can make life so much easier, but if you don't have
one, make your point even which is not so convencing, and move on.

solved,
acetone

【在 s****9 的大作中提到】

: I understand it can be the problem of the antibody and will never be solved,
: but want to try as hard as possible.
: Could you suggest a few antigen retrieval method? Do you think 5min acetone
: fixation is enough to expose the antigen epitope?
: Very few background on the staining. Do you think it is worthy to try
: different blocking reagents?
: Thanks so much. Really appreciate your suggestions.
:
: or

s*y2011-04-18 07:04

9 楼

One reminder is that the antibody concentration for IHC has to be at least
10 or 100x more than what you use for WB

size
4

【在 s****9 的大作中提到】

: IHC stain一个ER上膜蛋白，自己做的多克隆抗体（rabbit），western效果很好，size
: 和报道的一直，KO tissue没有带。但是IHC一直不成功，IHC用的是frozen tissue，
: acetone fixation，fast blue development。
: 想请教:
: １。这种ER上的膜蛋白用什么方法fix比较好。我用cold acetone 5min，也试过一次4
: ％PFA。似乎效果都不好，和KO没有区别。
: ２。有什么方法可以提高信号强度。有成熟的tyramide protocol可以提供吗?

C*r2011-04-18 07:04

10 楼

It really depends on individual antibody per se, no necessary 'at least 10-
100x more' for every IHC antibody, IMHO.

【在 s******y 的大作中提到】

: One reminder is that the antibody concentration for IHC has to be at least
: 10 or 100x more than what you use for WB
:
: size
: 4

s*92011-04-18 07:04

11 楼

Thanks. I will have a try of PFA fixation with protein retrieval.
The purpose of the IHC is to in situ visualize the cell type which has the
enzyme expression(located on the ER membrane). My laser capture data
suggests that the enzyme selectively expressed on certain regions but that
regions contains many cell types. IP is not useful for me.
My concern is that the epitope is intramembrane and thus cannot expose out
after fixation.

t
if

【在 g****0 的大作中提到】

: I had an antibody which could only recognize the PFA fixed sample but not
: the one fixed by cold methanol. You may try the trick, but the point is
: there may not be "logic"enoughto figure out experimental problems, so don't
: limit yourself to any possibility if you really want to push it.
: Further, I think you should do IP or westernblotting on native gel to see if
: what the antibody recognize. Meanwhile, you can try stain the protein
: overexpressed in cell culture.
: I know a good antibody can make life so much easier, but if you don't have
: one, make your point even which is not so convencing, and move on.
: