Rhs 1

Liver tissues were fixed in 3.7% buffered formalin, and then embedded in paraffin wax. The samples were cut into 3-μm sections and stained with hematoxylin & eosin (H&E) and Sirius Red (Direct Red 80, Aldrich, Milwaukee, WI) to detect collagen deposition. For immunohistochemistry, serial sections were deparaffinized and hydrated through a graded alcohol series. Antigen retrieval was performed by heating the sample in 0.01 M citrate buffer (pH 6.0) using a microwave vacuum histoprocessor (RHS-1, Milestone, Bergamo, Italy) at a controlled final temperature of 121 °C for 15 min. To block endogenous peroxide activity, the sections were quenched in 3% hydrogen peroxide in methanol and then blocked with 1% bovine serum albumin in PBS. Sections were incubated with primary antibodies against α-SMA and HtrA2/Omi diluted 1:500 in Antibody Diluent (Golden Bridge, Mukilteo, WA) at 4 °C. After washing, the peroxidase EnVision System (HRP rabbit/Mouse Envision System TM, Dakocytomation, Denmark) was applied at room temperature for 5–10 min. Peroxidase activity was detected with 3,3′-diaminobenzidine tetrachloride (DakoCytomation) and hematoxylin counterstain (DakoCytomation). The percent staining was calculated by the software of the Optimas 6.5 system.

A 5-µm-thick cross-section of a paraffin-embedded block was moved to a silanized glass slide. The sections were then de-paraffinized using xylene and rehydrated using a series of graded alcohols. Antigen retrieval was performed using a microwave vacuum histoprocessor (RHS-1; Milestone, Bergamo, Italy) by heating samples in 0.01 M citrate buffer (pH 6.0) for 20 min to a final temperature of 121 °C. The section was incubated for 10 min with hydrogen peroxide (3%) in methanol to prevent endogenous peroxide activity. Slides were then incubated with anti-CD3 (Abcam), anti-CD68 (clone: KP1, Dako, Carpinteria, CA, USA), and anti-PD-L1 (clone: 22C3, Dako) antibodies. After washing, the EnVision+ system HRP-labelled polymer (Dako) was used at 24 °C for 5 min. The slides were treated with 3,3′-diaminobenzidine for 5 min and then counterstained with hematoxylin.

Immunocytochemical identification of the HPV L1 capsid protein in cervical cytology samples was performed using the Cytoactive^® HPV L1 screening set (Cytoimmun Diagnostics GmbH, Primasens, Germany) according to the manufacturer's protocol. The coverslip and mounting medium of Papanicolaou-stained cervical smear slides in xylene were separated, and the residual xylene on the slides was removed with alcohol. To retrieve the antigen, 0.01mol/L citrate buffer (pH 6.0) was used, and the sample was heated in a microwave vacuum histoprocessor (RHS-1; Milestone, Bergamo, Italy) at a temperature of 121°C for 15 minutes. Slides were kept at room temperature for 10 minutes to cool. Subsequently, one droplet of HPV L1 capsid antibody was applied to the cervical smear slides, and slides were incubated for 1 hour at room temperature. One drop of ready -to- use- chromogen solution (3-amino-9-ethylcarbazole) was applied to the slide for 5 minutes. Mayer's hematoxylin counterstain was then applied. At least 1 positive control slide supplied by the manufacturer was included in each batch. Immunocytochemistry was performed using the first specimen.