Nagaso H, Murata T, Day N, Yokoyama K K
Gene Engineering Division, BioResource Center, RIKEN (The Institute of Physical and Chemical Research), Tsukuba Science City, Ibaraki, Japan.
J Histochem Cytochem. 2001 Sep;49(9):1177-82. doi: 10.1177/002215540104900911.
Proteinase K is widely used in methods for detection of transcripts in biological specimens by in situ hybridization (ISH). However, treatment with proteinase K hampers detection of RNA and protein simultaneously. We have developed a method for double staining of transcripts and proteins by ISH and IHC staining in imaginal discs and embryos of Drosophila. Instead of treatment with proteinase K, samples are treated with ethanol plus xylene and with acetone. Acetone renders cell membranes permeable to probes and antibodies without damaging tissue integrity, whereas treatment with proteinase K sometimes damages tissues. Treatment of samples with acetone allows hybridization of probe with transcripts in tissue. It is also effective for immunological staining of samples after ISH with a riboprobe. Thus, our method allows detection not only of transcripts but also of specific proteins in relatively intact single samples. (J Histochem Cytochem 49:1177-1182, 2001)
蛋白酶K广泛应用于通过原位杂交（ISH）检测生物标本中转录本的方法中。然而，用蛋白酶K处理会妨碍同时检测RNA和蛋白质。我们开发了一种在果蝇成虫盘和胚胎中通过ISH和免疫组化（IHC）染色对转录本和蛋白质进行双重染色的方法。样品不用蛋白酶K处理，而是用乙醇加二甲苯以及丙酮处理。丙酮使细胞膜对探针和抗体具有通透性，同时不会损害组织完整性，而用蛋白酶K处理有时会损害组织。用丙酮处理样品可使探针与组织中的转录本杂交。在用核糖探针进行ISH后，它对样品的免疫染色也有效。因此，我们的方法不仅可以检测相对完整的单个样品中的转录本，还可以检测特定蛋白质。（《组织化学与细胞化学杂志》49:1177 - 1182, 2001）
