Hematoxylin and eosin h e

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.

Histopathology evaluation using hematoxylin and eosin (H&E) staining kits (Vector Laboratories, Newark, CA) of the lungs, liver, kidney, and some cases of metastasized tumor were carried out. The excised frozen organs/tissue were embedded in optimum cutting temperature (OCT) compound and processed in a cryostat (Leica CM3050 S Research Cryostat, Leica Biosystems Inc., Buffalo Grove, IL, USA). Five micromoters thicknesses of each of the tissue an organs were sectioned along the longitudinal axis using a comparable technique from our recent studies [64 (link), 65 (link), 71 (link)]. These sections were placed on glass slides and allowed to stay overnight in the −80 °C freezer. Prior to the H&E staining, the samples on the glass slides are allowed to thaw and are fixed in 80% Methanol at 4 °C for 5 min, PBS for 10 min, rinsed in deionized water. The resulting samples were then stained with hematoxylin and eosin (H&E) following the methods as recommended by Vector Laboratories. The stained slides were examined using a 20 × objective lens TS100F Nikon light microscope (Nikon Instruments Inc., Melville, NY, USA) that was coupled with a DS-Fi3 C digital camera.