Goat poly

The histopathological analysis of ECPU-0001 was done by IHC. Briefly, sacrificed tumor samples were fixed and embedded in a wax block. From each paraffin-embedded sample, sections were cut in 5 μm by a microtome. Then deparaffinized and rehydrated were done in different percentage of xylene and alcohol respectively. Slides were treated and antigen was retrieved with 3% of hydrogen peroxide in methanol. Slides were blocked and exposed with rabbit polyclonal antibodies for either BCL-2 (diluted, 1:300; Mouse, Cell Signaling, Danvers, MA, USA), Bcl-XL (diluted, 1:500; Mouse, Santa Cruz) or Ki67 (diluted, 1:400; Goat poly, Santa Cruz) or BAX (diluted, 1:500; Mouse, Santa Cruz). Protein expression was observed by biotinylated goat anti-rabbit, anti-mouse IgG (Vector Laboratories, Burlingame, CA, USA). The diaminobenzidine (Vector Laboratories) was used as a substrate, which shows an insoluble brown precipitate. Nuclei were counterstained with Mayer’s Hematoxylin blue. Localized proteins images were captured and processed by the Multistain Imaging Module for BCL-2, BAX, Ki67 and Bcl-XL.

The efficacy of BSM-0004 and oncogenic expression of tumour marker Ki67 and hCA-IX were evaluated by immunohistochemistry of tumour samples as described in the previous study^51–54 (link). Briefly, tumour tissues were fixed with 4% paraformaldehyde then embedded with high grade of paraffin. The tissue-sections were cut in 5 µm thick using a microtome (Leica RM2255 Fully Automated Rotary Microtome, Wetzlar, Germany), then sliced tissues were fixed onto glass slides and deparaffinised with various percentage of xylene subjected to rehydrated with different concentrations of alcohol (C₂H₅OH). All slides were incubated with methanol containing 3% of hydrogen peroxide to be retrieved. After blocking with 5% horse serum, the slides were incubated with rabbit polyclonal antibodies for Ki67 (diluted, 1:400; Goat poly, Santa Cruz, Santa Cruz, CA) and anti-CA-IX (1:500 diluted, Mouse, abcam ab107257, Cambridge, UK). The immunohistopathological expression of Ki67 and hCA-IX was counter stained with biotinylated anti-rabbit and anti-mouse IgG (Vector Laboratories, Burlingame, CA). The diaminobenzidine (DAB) substrate was used to develop and precipitate the protein. Lastly, Mayer’s haematoxylin blue staining was performed to distinctly observe nuclear portion. The Ki67 and hCA-IX protein levels were detected using Leica microscope system (Wetzlar, Germany).