Trichrome stain kit

The histological observation was performed by staining with hematoxylin/eosin (H&E), Giemsa, and trichrome stains using paraffin sections. For H&E staining, paraffin-embedded sample slides were de-paraffinized, hydrated, and then stained with hematoxylin for 1 min. After rinse, the slides were stained with eosin for 5 min, rinsed, and sealed with cover slips. Tissue sections from tumor mass, kidneys, spleen, liver, heart, and lungs were used. The slides were counterstained with hematoxylin and mounted. To determine the effect of CPT on expression of Ki67, PCNA, Caspase-3, Caspase-8, Caspase-9, Bcl-2, Bax, Bid, Bad, Drp1, Opa1, Mfn1, and Mfn2 by immunohistochemistry, the slides were blocked in 5% bovine serum for 15 min, followed by incubation with the primary antibody at 4 °C overnight in a moist chamber. The sections were then incubated with the corresponding secondary antibodies. The antigen-antibody complex was detected by Dako Liquid DAB + Substrate-Chromogen System (Dako, Carpinteria, CA). All slides were examined under light microscopy. Giemsa stain and trichrome stain were performed according to the manufacturer’s instruction of Giemsa Stain Kit (ab150670) and Trichrome Stain Kit (ab150686) from Abcam (UK). All slides were examined under light microscopy.

The diabetes inducing drug, Streptozotocin was purchased from Abcam (ab 142155, Cambridge, UK), 6-gingerol (G1045) was purchased Sigma Aldrich, St. Louis, MO, USA. Antioxidant enzymes like superoxide dismutase (SOD) (ab 65354), Glutathione-S-transferase (GST) (ab 65326), catalase (CAT) (ab 83464) and Trichrome Stain Kit (Connective Tissue Stain) (ab150686) were bought from Abcam, Cambridge, UK. ELISA kits for the assay of inflammation by C-reactive protein (ab 108827), TNF-α (ab 46070), IL-1β (ab 100768), and IL-6 (ab 100772) were procured from Abcam, Cambridge, UK. Primary antibodies (TNF-α) and Goat Anti-Mouse IgG H&L (HRP) (abcam, Cambridge, UK) used in this study were purchased from Abcam, Cambridge, UK. All supplementary chemicals used in this study were of high purity grade obtained from commercial sources.

The lung tissues were fixed in 10% formalin and embedded in paraffin, and then were cut at 4 μm thickness for preparation of serial section slides. Tissue sections were stained with hematoxylin & eosin (H&E) for histological examination or subjected to immunohistochemical analysis with each of the indicated primary antibodies according to standard techniques. Three independent visual fields were examined for quantitative analysis using ImageJ 1.50e with an IHC Toolbox plugin according to the instructions (https://imagej.nih.gov/ij/plugins/ihc-toolbox/index.html). Trichrome Stain Kit (Abcam) was used for Masson’s trichrome stain in line with the manufacturer’s protocols. For analysis of calcium deposits, tissue sections were subjected to Von Kossa stain using Von Kossa Stain Kit (Abcam) according to manufacturer’s instructions. The histological and immunohistochemical analysis were performed by two trained technologists and verified by a pathologist.