Rabbit on rodent hrp polymer rmr622h

Formalin‐fixed, paraffin‐embedded tissue sections were deparaffinized in xylene and rehydrated through graded ethanol. Endogenous peroxidase activity was blocked with 3% H₂O₂ in methanol followed by antigen retrieval using Tris‐EDTA (pH 9) and blocking with Dako serum‐free protein block (X0909; Dako) or Background Sniper (BS966L; Biocare Medical). Immunostaining was performed using primary antibodies targeting ATP‐binding cassette sub‐family B member 1 (ABCB1) (C219; BioLegend), AR (N‐20; Santa Cruz Biotechnology), or AR‐V7 (31‐1109‐00; RevMAb Biosciences) and the Envision horseradish peroxidase (HRP) Rabbit detection system (K4003; Dako) or the Rabbit‐on‐Rodent HRP‐Polymer (RMR622H, Biocare Medical) with 3,3′‐diaminobenzidine as chromogen and counterstaining with haematoxylin. Sections were scanned using the Pannoramic 250 FLASH scanner and evaluated with the Pannoramic viewer 1.15.2 software (3D HISTECH).

Livers were fixed in 10% formalin overnight at 4°C, then gradually dehydrated with ethanol and embedded in paraffin. Immunohistochemistry and hematoxylin and eosin (H&E) staining were performed by the Texas Digestive Diseases Morphology Core at Baylor College of Medicine, as previously described.^{17 (link)} The following primary antibodies were used for immunohistochemistry: rabbit anti 2A peptide (1:7500, ABS31, Sigma-Aldrich), rabbit anti Ki67 (1:60, CRM325, Biocare); rabbit anti FAH (1:65, ab151998, Abcam); rabbit anti Cd34 (1:250, ab81289, Abcam). The Ki67, FAH and Cd34 antibodies were then detected with a Rabbit-on-Rodent HRP-Polymer (RMR622H, Biocare) and visualized with DAB chromogen (DB801, Biocare). The 2A antibody was detected and visualized using a Leica Bond Polymer Detection kit (DS9800). Tunel staining was performed and detected using ApopTag Peroxidase In Situ Apoptosis Detection Kit (Millipore, S7100). Reticulin staining was performed using the Epredia Reticulin Sliver Stain Kit (87025, Epredia), following the manufacturer instructions. Slides were counterstained with hematoxylin, dehydrated, and mounted with a permanent mounting medium. A Nikon Ci-L bright field microscope was used for imaging at the Integrated Microscopy core (Baylor College of Medicine).