Horseradish peroxidase conjugated streptavidin biotin complex

To analyze the expression of ABCA1, α-smooth muscle actin (SMA), and CD56 in carotid stenosis artery tissues, we used ABCA1 (Novus, Centennial, CO, USA), α-SMA (Dako, Santa Clara, CA, USA), and CD56 (Abcam, Cambridge, UK) antibodies, respectively. The arterial sections were incubated with 3% H₂O₂ in methanol to block endogenous peroxidase activity. After antigen retrieval, the slides were incubated with an anti-ABCA1, α-SMA, CD56 antibodies diluted at 1:100, 1:400, and 1:100 respectively at 4 °C overnight, and then incubated for 1 h with a biotinylated secondary anti-rabbit and mouse antibody at room temperature (RT). The sections were incubated with a horseradish peroxidase-conjugated streptavidin-biotin complex (Dako) and 3,3-diaminobenzidine (EnVision Systems, Santa Clara, CA, USA) was used to visualize the chromatic signals. Images were taken using a digital slide scanner (3DHISTECH Ltd., Budapest, Hungary). Finally, the percentage of positive signals was quantified in all sections by 3DHISTECH Ltd. QuantCenter 2.2 program (3DHISTECH Ltd.).

To observe the degree of hepatocyte proliferation following transplantation with MSCs or the control, BDL rat liver tissues were stained with anti-PCNA (Santa CruzBiotechnology, Dallas, Texas, USA) using immunohistochemistry. The liver tissues were embedded in paraffin and sectioned. The sectioned tissues were incubated in 3% H₂O₂ in methanol to block endogenous peroxidase activity. After antigen retrieval, the tissues were incubated with a primary antibody (1:200) at 4 °C overnight, followed by a 1-h incubation with biotinylated secondary anti-rabbit antibody at room temperature. Incubation with horseradish peroxidase-conjugated streptavidin–biotin complex (DAKO, Santa Clara, CA, USA) and 3,3-diaminobenzidine (EnVision Systems, Santa Clara, CA, USA) was performed to generate a chromatic signal. The samples were counterstained with Mayer’s hematoxylin (DAKO). Additionally, the percentage of hepatocytes with PCNA-positive nuclei relative to the total number of hepatocytes was calculated in randomly selected sections using a digital slide scanner (3DHISTECHLtd., Budapest, Hungary). The experiment was analyzed in at least triplicate.

To analyze the degree of hepatocyte proliferation in tissues following treatment with WKYMVm or no treatment, we used an anti-proliferating cell nuclear antigen (PCNA; Santa Cruz Biotechnology, Dallas, TX, USA) antibody. The liver sections were incubated with 3% H₂O₂ in methanol to block endogenous peroxidase activity. After antigen retrieval, the slides were incubated with an anti-PCNA antibody diluted 1:200 at 4 °C overnight, and then incubated for 30 min with a biotinylated secondary anti-rabbit antibody at room temperature (RT). The sections were incubated with a horseradish peroxidase-conjugated streptavidin-biotin complex (Dako, Santa Clara, CA, USA) and 3,3-diaminobenzidine (EnVision Systems, Santa Clara, CA, USA) to visualize the chromatic signals. Images were taken using a digital slide scanner (3DHISTECH Ltd., Budapest, Hungary). Finally, the percentage of PCNA-positive hepatocytes was quantified in randomly selected sections (three fields per group at 400× magnification).