作者pent (愛、失敗、死亡)
看板Biotech
標題[求救] IHC的detergent
時間Sat Jan 26 22:10:35 2013
用的是paraffin section 的心臟組織,formalin fix overnight, MI後過3天
要看的是CD45, BD pharmaco.賣的, 理論上MI後過3天應該看的到白血球進入心臟?
但是我一直染不出來
protocol是實驗室的學長已經試過的, 不過他的組織是血管
猜測:
我們lab做IHC都會用PAP pen, 畫一層防水的臘, 畫在組織周圍
方便將solution, 與組織反應
但是, 如果detergent是用triton x-100的話
在反應的過程中, 他就很容易流出來,
隔天去看發現組織都乾掉,
做不出來了
但是, 有人跟我說detergent可以幫助Ab進入細胞, 尤其是有需要進到細胞核的染色
So
想請問一下, 1抗, 2抗需不需要泡在帶有detergent的BSA in PBS or TBS裡面??????????
Thx
查到的: from IHC world
Do I need detergent to permeabilize the cells?
Answer:
Once cells are fixed (in suspension, smeared on a slide, cytospun, inside a
piece of tissue) they no longer can select intracellular traffic of molecules
. In addition, processes such as drying and lipid solvent treatment (acetone,
ethanol etc.) creates huge holes in the subcellular structure.
Antibodies can easily cross cell membranes (cytoplasmic, nuclear) in fixed
cells.
Whether they can reach the antigen in case of fixation dependent antigen
masking, is another problem. No detergent is able to do antigen unmasking.
The crucial role of low levels of detergents such as Tween 20 is to reduce
surface tension in washing solutions, allowing the slides to remain wet.
There is no role for detergent in permeabilization of fixed cells. Only
exceptions are:
* saponin treatment of living cells for flow cytometry staining
* high concentration detergent mediated extraction of nuclear / cellular
matrix-associated substances in lightly fixed cells.
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◆ From: 140.109.40.72
1F：推 caesar0926:我是PBS+tween20 wash + 配抗體 01/27 01:38
2F：→ caesar0926:在用parapen畫範圍之前我會先用DDH2O在浸一下片子 01/27 01:40
3F：→ caesar0926:而且會將想畫的範圍吸乾再畫 不然很容易有破洞 01/27 01:41
4F：→ caesar0926:我之前有直接PBST wash後直接畫發現根本就會被溶掉 01/27 01:42
5F：→ caesar0926:另外 假設你是4度c O/N 有加一點水在放片子的盒子嗎? 01/27 01:43
6F：→ caesar0926:不然很容易乾掉 01/27 01:44
7F：→ caesar0926:還有可以不用parapen 應該也可以染 01/27 01:45
8F：→ caesar0926:在 blocking 後 你後面加上的抗體 大多都可以被限制在 01/27 01:45
9F：→ caesar0926:BLOCKING 時的範圍內 01/27 01:46
10F：推 goshr:用一小張parafilm蓋在玻片上, 避免乾掉 01/27 10:03
11F：→ goshr:其實, 不用overnight啦, 一抗會上就上了, 太久沒好處 01/27 10:04
12F：→ pent:我是放在wet chamber裡的 01/27 18:43
13F：→ pent:但, 不同的detergent真的有差嗎? 01/27 18:43
14F：→ pent:1F大大, 不管啥抗體都是用tw-20嗎? 01/27 18:43
15F：推 caesar0926:是啊 01/28 08:03
16F：推 Dauthi:我們實驗室和goshr使用的一樣 蓋個parafilm就好了 01/28 09:49