[求救] IHC的detergent

看板Biotech (生命科學)作者pent (愛、失敗、死亡)時間13年前 (2013/01/26 22:10)推噓4(4推 0噓 12→)

留言16則, 4人參與討論串1/1

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. -- ※ 發信站: 批踢踢實業坊(ptt.cc) ◆ From: 140.109.40.72

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caesar0926

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