Dm 4000b photomicroscope

Tissues were obtained, fixed and embedded with paraffin according to standard procedures. Anti-SMYD1 (1:200) and anti-SRF (1:500) were used to stain limb bud vessels in 5-μm sections. After staining with DAB (brown precipitate). Slides were counterstained with hematoxylin. A Leica DM 4000B photomicroscope was used to take the images.

Tumor and mouse tissue specimens were fixed in 4% formaldehyde for 12 h, processed, and embedded in paraffin blocks. Sections (4 μm) of the lungs and other tissues were stained with hematoxylin and eosin (H&E). Histopathological changes were observed under a light microscope. For immunohistochemical staining, sections were cut from the paraffin blocks and incubated with anti-Ki-67 (1:250) (Abcam, MA, USA), and anti-AR (1:50; N-20) (Santa Cruz, CA, USA) as primary antibodies. Immuno-reactivity was visualized using peroxidase-DAB22 (DAKO, Japan). Three tumors per group were analyzed. The number of Ki67-positive cells and AR-positive cells was quantified using DM 4000B photomicroscope (Leica, DE), and the apoptotic index in six random fields per group was counted.

After 15 days, the mice were euthanized, and the melanoma tumors were removed and fixed with 4% polyformaldehyde. Immunohistochemical (IHC) reactions were performed using the streptavidin peroxidase method. For the negative control, the slide was incubated in PBS. Primary antibodies against CD31 (ab28364), Ki-67 (ab15580), CD8a (ab4055), and Neuropilin 1 (ab81321) were applied according to the staining kit protocol. Biotinylated goat anti-rabbit and anti-rat antibodies (BOSTER SA1022 or SA1025) were used as the secondary antibodies. Images were recorded using a Leica DM 4000B photomicroscope. Image-Pro Plus 6.0 software (Media Cybernetics) was used to analyze the mean integrated optical density (mean IOD) of the target protein expression in tumor sections according to the following formula: mean IOD = IOD/area of the tumor section. Areas of necrosis and staining artifacts were excluded by AOI tools. Samples were stained from the tumor tissues of two to three mice per group and 3 to 4 slides by manual immunohistochemical (IHC) staining. Three images of each slide were assessed.

Tumour samples obtained from in vivo studies were rinsed in PBS and fixed in 10% paraformaldehyde/PBS. Samples were dehydrated in 70% ethanol, paraffin‐embedded and sectioned (4 μm). Deparaffinized sections were stained for E‐cadherin, Vimentin, Snail+Slug, MMP2 and Smad4 antigens. Briefly, samples were rehydrated with ethanol. Tissue sections were then pre‐incubated with 10% normal goat serum in PBS (pH 7.5) followed with incubation with primary antibody overnight at 4°C. Tissue sections were then stained with biotinylated secondary antibody (Vector Laboratories, Burlingame, California, USA) for 1 hr at room temperature, followed by the Vectastain Elite ABC reagent (Vector Laboratories, Burlingame, California, USA) for 30 min. The peroxidase reaction was developed with diaminobenzidine (DAB kit; Vector Laboratories, Burlingame, California, USA), and the slides were counterstained with haematoxylin. Images were taken using a Leica DM 4000B photo microscope (magnification, ×200). Staining intensity was scored as 0 (negative), 1 (weak), 2 (medium) and 3 (strong). Extent of staining was scored as 0 (0%), 1 (1–25%), 2 (26–50%), 3 (51–75%) and 4 (76–100%), according to the percentage of the whole carcinoma area which was positively stained with each antibody. The sum of the intensity score and the extent score was used as the final staining score.