Multiplex IHC Analysis of Liver Tissue

Multiplex immunohistochemistry staining (mIHC) was performed to analyze the expression of various factors in liver tissue. Deparaffinized and hydrated tissue sections were boiled in sodium citrate buffer (pH 6.0) for antigen retrieval. mIHC was performed using a four-color immunohistochemistry staining kit (PANOVUE, 10079100020) according to the manufacturer’s protocol. Next, α-SMA antibody (1:400 dilution, #19245, CST) was incubated overnight and washed with TBS. Subsequently, 30 μL of proportionally diluted tyramide signal amplification fluorescent solution (1:100) was incubated at room temperature for 20 min. Then, the PPARγ antibody (1:300 dilution, ab59256, Abcam) was also processed following the same conditions and steps. The stained slides were scanned and quantified using the HALO Highplex FL (Indica Labs; Albuquerque, NM) analysis module of Halo software (Erber et al., 2021 (link)).