Nis elements viewer 3

Human prostate were embedded in Tissue-Tec OCT compound (SakuraFinetek Japan, Tokyo, Japan) and snap frozen. Then tissue was sectioned in 10 μM thick slices and thaw, mounted onto glass slides using a cryostat (Leica CM 1850, Wetzlar, Germany), air-dried, and fixed for 10 min in ice-cold acetone. Slides were washed in PBS and then incubated for 2 h in a mixture of PBS supplemented with 0.2% [v/v] Triton X-100 and 0.1% [w/v] bovine serum albumin, followed by incubation overnight with the primary antibody (rabbit polyclonal to PDE5A, 1:100) and antibody mixture of the PDE5A antibody (1:100) and goat polyclonal to nNOS (1:50, Abcam, ab1376 Cambridge, UK). The secondary antibodies employed to visualize the localization of the two primary antibodies (Jackson ImmunoResearch Inc. West Grove, PA, USA) were Cy3-conjugated goat anti-rabbit IgG (1:1000) and Cy2-conjugated donkey anti-goat IgG (1:400). DAPI was used for staining the nucleus. Negative controls were performed for all samples by omitting the primary antibodies. Human lung tissue was used as a positive control for PDE5A staining. Visualization was done with a laser microscope (Olympus, Tokyo, Japan). Colocalization analysis was performed using NIS-Elements Viewer 3.20 (Nikon, Japan).
All experimental protocol were approved by the research committee of Wuhan University.

Human prostate tissues were embedded in Tissue-Tec OCT compound (Sakura Finetek Japan, Tokyo, Japan) and snap frozen. The tissues were then sectioned into 10 μM slices and then thawed, mounted onto glass slides using a cryostat (Leica CM 1850, Wetzlar, Germany), air-dried, and fixed for 10 min in ice-cold acetone. Slides were washed in phosphate-buffered saline (PBS) and then incubated for 2 h in a mixture of PBS supplemented with 0.2% [v/v] Triton X-100 and 0.1% [w/v] bovine serum albumin, followed by overnight incubation with the primary antibody (Table 2). The secondary antibodies employed to visualize the localization of the primary antibodies (Jackson ImmunoResearch Inc., West Grove, PA, USA) were Cy3-conjugated goat antirabbit IgG (1:1000) and Cy2-conjugated donkey antigoat IgG (1:400), and DAPI was used to stain the nucleus. Colocalization analysis was performed using NIS-Elements Viewer 3.20 (Nikon, Japan). Visualization was performed with a laser microscope (Olympus, Tokyo, Japan).

Rat CC were embedded in Tissue‐Tec OCT compound (SakuraFinetek Japan) and snap‐frozen. Then, the tissue was sectioned into 10 μm thick slices, thawed and then mounted onto glass slides using a cryostat (Leica CM 1850). After being air‐dried, sections were fixed for 10 minutes in ice‐cold acetone and washed in PBS. Then, sections were incubated for 2 hours in a mixture of PBS supplemented with 0.2% Triton X‐100 and 0.1% bovine serum albumin. After incubation overnight with the primary antibody mixture of SMMHC (smooth muscle myosin heavy chain, mouse polyclonal to MYH11 [Myosin Heavy Chain 11], 1:100) and PDE5 antibody (rabbit polyclonal to PDE5A, 1:100), the secondary antibodies (Jackson ImmunoResearch Inc) labelled with FITC‐conjugated anti‐mouse IgG (1:200) and Cy3‐conjugated anti‐rabbit IgG (1:1000) were used to visualize the localization of the two primary antibodies. DAPI was used for staining the nucleus. Negative controls were performed for all samples by omitting the primary antibodies. Rat lung tissue was used as a positive control for PDE5A staining. Stained sections were viewed by a laser microscope (Olympus). Analysis was performed using the NIS‐Elements Viewer 3.20 (Nikon).