4 6 diamidino 2 phenylindole or dapi

Immunohistochemistry staining against ROCK subtypes 1 and 2 and α smooth muscle actin (αSMA) was performed on corpus cavernosum of an ED patient. Samples were fixed in 10% neutral-buffered formalin and embedded in paraffin. Paraffin sections were dewaxed in xylene and rehydrated with a graded ethanol series. Slides were washed in Tris-buffered saline (TBS). For epitope retrieval, slides were exposed in citrate buffer (pH 6) and heated to about 90°C for 60 minutes. After washing in TBS, the slides were blocked for 60 minutes using BSA 1%, NGS 3%, Triton X-100 0.3% in PBS (Sigma-Aldrich, St. Louis, MO, USA), followed by incubation overnight on 4°C in a humidified chamber with 1/200 Rb ROCK1 (Abcam, Cambridge, UK), 1/200 Rb anti-ROCK2 (Abcam, Cambridge, UK), 1/200 Ms anti-smooth muscle actin (Dako, Copenhagen, Denmark). Then, slides were rinsed in PBS, incubated for one hour on room temperature with secondary antibodies 1/500 Alexa Fluor 594 donkey anti-mouse, 1/500 Alexa Fluor 488 donkey anti-rabbit (Invitrogen, Carlsbad, CA, USA) as secondary antibodies. Control staining in the absence of primary antibodies was used to evaluate nonspecific staining by secondary antibodies. Nuclear staining was performed using 4',6-diamidino-2-phenylindole or DAPI (Invitrogen, Carlsbad, CA, USA). Slides were evaluated and photographed using a Olympus microscope (Olympus, Tokyo, Japan).