Citric acid ph 6

For immunohistochemistry staining, the liver tissues were first fixed in a solution with 4% paraformaldehyde; they were then embedded in paraffin. Sections of 4 μm thickness were then cut from these paraffin-embedded tissues. The antigen repair solution with citric acid (pH 6.0, Service Bio, China) was employed to enhance antigen retrieval. After this step, the slides were treated with a 10% rabbit serum (Service Bio) to block non-specific binding. The slides were placed in primary antibody (Table 2) and stored at 4°C overnight. Following this, the slices were subjected to DAB (3,3′-diaminobenzidine) treatment. Subsequently, the slides were dehydrated using alcohol and subjected to hematoxylin (Service Bio, China) staining. The stained slides were examined under a microscope (Nikon E-100 Tokyo, Japan). Images were captured using the Case Viewer program (3DHISTECH, Hungary Ltd.).

Immunohistochemistry was used to detect the Bcl-2, Bax, and cleaved caspase-3 positive cells in the ischemic cortical area after 24 h of reperfusion. Paraffin sections were dewaxed and hydrated in graded concentration of ethanol and xylene; antigen retrieval was conducted in citric acid (pH 6.0) (Servicebio, China). Sections were incubated with 3% hydrogen peroxide for 10 min to block endogenous peroxidase and added with 1% BSA (Servicebio, China) for 30 min and then mixed with monoclonal antibody (rabbit anti-mouse Bcl-2, dilution of 1:100; rabbit anti-mouse Bax, dilution of 1:200 rabbit anti-mouse cleaved caspase-3, dilution of 1:300. All from Servicebio, China) overnight at 4°C. Next, sections were mixed with polymerase for 20 min and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG monoclonal antibody (dilution of 1:200, Servicebio, China) for 1 h at room temperature. DAB (Servicebio, China) was used as chromogen and hematoxylin as counterstain after a rinse in distilled water, section dehydrated, and coverslipped. The expression level of Bcl-2, Bax, and cleaved caspase-3 was calculated as number of positive cells/total cells number. The observers who recorded measured results were blind to the experimental design.