作者pent (爱、失败、死亡)
看板Biotech
标题[求救] IHC的detergent
时间Sat Jan 26 22:10:35 2013
用的是paraffin section 的心脏组织,formalin fix overnight, MI後过3天
要看的是CD45, BD pharmaco.卖的, 理论上MI後过3天应该看的到白血球进入心脏?
但是我一直染不出来
protocol是实验室的学长已经试过的, 不过他的组织是血管
猜测:
我们lab做IHC都会用PAP pen, 画一层防水的腊, 画在组织周围
方便将solution, 与组织反应
但是, 如果detergent是用triton x-100的话
在反应的过程中, 他就很容易流出来,
隔天去看发现组织都乾掉,
做不出来了
但是, 有人跟我说detergent可以帮助Ab进入细胞, 尤其是有需要进到细胞核的染色
So
想请问一下, 1抗, 2抗需不需要泡在带有detergent的BSA in PBS or TBS里面??????????
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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※ 发信站: 批踢踢实业坊(ptt.cc)
◆ From: 140.109.40.72
1F：推 caesar0926:我是PBS+tween20 wash + 配抗体 01/27 01:38
2F：→ caesar0926:在用parapen画范围之前我会先用DDH2O在浸一下片子 01/27 01:40
3F：→ caesar0926:而且会将想画的范围吸乾再画 不然很容易有破洞 01/27 01:41
4F：→ caesar0926:我之前有直接PBST wash後直接画发现根本就会被溶掉 01/27 01:42
5F：→ caesar0926:另外 假设你是4度c O/N 有加一点水在放片子的盒子吗? 01/27 01:43
6F：→ caesar0926:不然很容易乾掉 01/27 01:44
7F：→ caesar0926:还有可以不用parapen 应该也可以染 01/27 01:45
8F：→ caesar0926:在 blocking 後 你後面加上的抗体 大多都可以被限制在 01/27 01:45
9F：→ caesar0926:BLOCKING 时的范围内 01/27 01:46
10F：推 goshr:用一小张parafilm盖在玻片上, 避免乾掉 01/27 10:03
11F：→ goshr:其实, 不用overnight啦, 一抗会上就上了, 太久没好处 01/27 10:04
12F：→ pent:我是放在wet chamber里的 01/27 18:43
13F：→ pent:但, 不同的detergent真的有差吗? 01/27 18:43
14F：→ pent:1F大大, 不管啥抗体都是用tw-20吗? 01/27 18:43
15F：推 caesar0926:是啊 01/28 08:03
16F：推 Dauthi:我们实验室和goshr使用的一样 盖个parafilm就好了 01/28 09:49