Masson s trichrome

All the kidney tissue samples were fixed in neutral formalin and paraffin embedded block was made. After thin sectioning (5 µm), the sections were mounted on slides. The hematoxylin and eosin (H&E) staining was performed to examine the architecture of kidney tissues. Histopathological examination was performed by using a light microscope (Olympus, Tokyo, Japan) and the slide pictures were shot accordingly. The histological findings were performed by double-blinded histopathologist. Furthermore, Masson’s trichrome was used to evaluate the morphology of fibrosis as per the manufacturer’s protocol (Abcam, UK).

Tumor tissues were formalin-fixed (VWR, Radnor, PA, USA) and embedded in paraffin. Six-µm sections were prepared for Masson’s trichrome (Abcam) and hematoxylin and eosin (H&E) (vector laboratories) as per the manufacturer’s protocols. Images for histology and trichrome staining were taken using an EVOS microscope. Masson’s trichrome was quantified using user-defined macro in ImageJ 1.51j8 software, National Institutes of Health, USA.
Post-treatment tumor tissue was collected, and protein lysates were obtained for western blot analysis. Briefly, the protein lysate was processed on SDS-polyacrylamide gel electrophoresis and transferred to the nitrocellulose membrane. Membranes were then incubated overnight at 4 °C with mouse anti-α-SMA antibody (1:1000), mouse anti-HIF1A antibody (1:1000), mouse anti-vimentin antibody, and mouse anti-GAPDH antibody (1:5000) followed by incubation with secondary antibody anti-rabbit 800 (1:25,000) (LI-COR Odyssey, Lincoln, Nebraska USA). A LI-COR Odyssey Infrared Imaging System Model 912 was used to visualize the protein bands, then calculated based on densitometry normalized to the GAPDH band using ImageJ 1.51j8 software, National Institutes of Health, USA. All densitometry calculations represent an average of three biological replicates.

The collagen content in diabetic wounds was stained with Masson’s Trichrome (Abcam) and the blue intensity was quantified using ImageJ software as previously described. Briefly, images were processed using color deconvolution to extract blue color, which was identified as collagen. The fixed threshold was then applied to each image and the intensity of blue color was analyzed.

Paraffin blocks and tissue specimens from UVB-irradiated dorsal skin tissue of mice were prepared by modifying a previously reported method [19 (link)]. The tissue sections were stained using three staining reagents, namely H&E (Sigma-Aldrich, St. Louis, MO, USA), Masson’s trichrome (Abcam, Cambridge, UK), and toluidine blue (Sigma-Aldrich) to analyze the microfold number, epidermal thickness, inflammatory and mast cell counts, and collagen fiber-occupied region areas, respectively. Histomorphometry was performed using an Eclipse 80i microscope (Nikon, Tokyo, Japan) equipped with iSolution FL (Ver. 9.1) software (iSolutions Inc., Burnaby, BC, Canada).