Xylene

The invariable IHC procedures for all protocols included sectioning FFPE tissues at 5 μm and placing them on positively charged glass slides. The slides were soaked in xylene (Fischer Scientific, Pittsburg, PA, USA) three times for 5 min to remove paraffin. These sections were then rehydrated through a gradient of ethanol (Fischer Scientific) for 5 min in each concentration, 100%, 100%, 95%, and 70% ethanol, followed by de-ionized water. In order to reduce the volume of the reagents tested and liquid loss, tissues were encircled with a hydrophobic barrier pen (ImmEdge™ Pen, Ted Pella Inc., Redding, CA, USA).

Immunohistochemistry was performed using the IHC Staining Kit (Dako, Santa Clara, CA, USA). Tissue sections were deparaffinized in xylene (Thermo), hydrated in phosphate‐buffered saline (Welgene), and blocked with Background Reducing Solution (Dako). The sections were incubated with anti‐CASQ2 (#NBP1‐87304; NOVUS), anti‐aSMA (#BS70000; Bioworld Technology), anti‐FSP1 (#BS7671; Bioworld Technology), anti‐HIF1α (#NB100‐131; NOVUS), anti‐vimentin (#5741; Cell Signaling Technology), anti‐pan‐cytokeratin (#M3515; Dako), and anti‐ki67 (#9027; Cell Signal Technology) at 4 °C overnight, followed by incubation with horseradish peroxidase‐conjugated anti‐secondary antibody (Dako). The signal was developed using diaminobenzidine and hydrogen peroxide, resulting in a brown precipitate. The sections were counterstained with hematoxylin (Dako), dehydrated, and mounted. The DAB area was quantified using IHC Toolbox in ImageJ software (NIH) with 20 random histological fields from five slides of tumor tissues per group [22 (link)].

The nephrotoxic effects of D. russelii and D. palaestinae venoms were assessed by injecting 10 μg of the venom into the caudal vein of 4 male CD-1 mice (18–22 g). Both kidneys were harvested immediately after the death of the animal and were washed with 1X PBS, fixed in 10% buffered formalin for 24 h, and dehydrated with ascending concentrations of ethyl alcohol (70 and 95% for 30 min; 100% for 2 h), and cleared in xylene (Thermofisher, Waltham, MA, USA). Tissues were embedded in paraffin (Thermofisher, Waltham, MA, USA) at 58 °C, following which 3 μm sections were prepared using a Leica microtome (RM2245, Wetzlar, Germany). The slides were then stained with hematoxylin (Leica, Wetzlar, Germany), eosin (Leica, Wetzlar, Germany) and Masson’s trichrome staining (MTS; Path Stains, Bengaluru, India). The slides obtained were visualised using an Olympus light microscope (Ix81, Olympus, Shinjuku, Japan) at a 40× magnification, and images were acquired and analysed using CellSens dimension imaging software (Olympus, Shinjuku, Japan). The histological structure of renal tubules and glomeruli of the treatment group (10 μg of venom) was assessed in comparison to the control that received 200 μL of normal saline [82 ].

Samples (gWAT, iWAT, and BAT) underwent fixation in 10% neutral buffered formalin (Millipore Sigma, CAT#HT501128) for 62 h. Following fixation, samples were transferred into 70% ethanol for future processing. Samples underwent dehydration via ethanol (1 × 90% 30 min, 3 × 100% 40 min) and xylene (Fischer Scientific) (3 × 45 min). Samples were embedded in paraffin and 10 µm sections were mounted on 1.2 mm Superfrost™ slides. Slides were stained with Harris hematoxylin and eosin (H&E), imaged using a Nikon Eclipse 80i microscope (CAT#PL-D655CU-CYL), and images were captured with Pixelink software. Three images from each animal (~150 cells/image) were sampled to determine cross-sectional area and percent multilocular (ImageJ software, National Institute of Mental Health, Bethesda, MD, USA).

The colon tissue was cut into 5-μm sections and placed on positively charged slides. Sections were rehydrated as follows: xylene (ThermoFisher Scientific) for 3 times, 100% FLEX (ThermoFisher Scientific) twice, 95% FLEX once, 70% FLEX once, 50% FLEX once with 3 min for each step. Mucins were stained as described previously [46 ]. Periodic acid Schiff’s reagent and Alcian blue were used to distinguish neutral (bright red magenta) and acidic (dark blue) mucins. Nuclear Fast Red was used to stain the cell nucleus (red) for better visualization of the tissue under a light microscope. Briefly, sections were stained in 0.05% Alcian blue (ThermoFisher Scientific), made in 3% acetic acid, for 15 min and then rinsed in running tap water (3 min) followed by distilled water (2 min). Sections were then stained with 0.05% periodic acid for 5 min, washed in running tap water for 3 min, and then rinsed in distilled water for 5 min. Next, sections were stained with the Schiff’s reagent (Ricca Chemical Company, Arlington, TX, USA) for 10 min, washed in running tap water for 3 min and then distilled water for 5 min. Finally, sections were stained with Nuclear Fast Red for 5 min followed by washing the sections in running tap water 3 min and in distilled water for 5 min. The sections were then allowed to dry and a cover slip was applied using Permount^® mounting medium (ThermoFisher Scientific).

Tissue samples for histopathological analyses and immunohistochemistry assays were processed following standard procedure as previously reported[22 (link)]. Briefly, formalin-fixed tissues were dehydrated in a series of increasing concentrations of ethanol (70%, 80%, 95% and 100%) before they were incubated in Xylene (Thermo Fisher Scientific) two times with each time incubating for 1 h at room temperature, and then infiltrating with melted paraffin wax in an oven at 65 °C. Paraffin-embedded tissue blocks were sectioned at 5 μm using a microtome. The sections were loaded to polylysine-coated glass slides, dried overnight at 42 °C, and stored at room temperature for further use.