Ab5694

Cells seeded on a glass coverslip were washed three times in warm PBS prior to fixation in 4% paraformaldehyde (PFA, 10 min, room temperature). Cells were subsequently permeabilized with 0.5% Triton-X-100 (10 min, room temperature), quenched with 3%H₂O₂ (30 min, room temperature), blocked with a protein block (Dako, 30 min, room temperature) and incubated overnight with primary antibodies (ACTA2 1:200, Abcam #ab5694; cleaved-caspase 3 1:200, Cell Signalling #9661; Ki67 1:500, Thermo Fisher, A7L6 HSPG2 1:300 (mAb targeting domain IV of mouse HSPG2, Merck Millipore; CCN1 1:1000 (anti-HSPG2 antibody, developed by the Whitelock laboratory) and t-RFP 1:100, Evrogen #AB233). Subsequently, secondary antibodies (Envision) coupled to HRP were applied and detection was performed with DAB.

Fibroblasts seeded on 8-well Lab-Tek II chamber glass slides (Nalgene Nunc International) were incubated in serum-free DMEM with or without tenascin-C (2 μg/ml) for 72 hours. Cells were then fixed, permeabilized, and incubated with antibodies against ASMA (Abcam, 1:500, ab5694), type I collagen (Southern Biotechnology, 1:100, 1310-01), RP105 (Abcam, 1:100, ab184956), and MD1 (Santa Cruz, 1:50, sc-390613), followed by Alexa Fluor–labeled secondary antibodies (Invitrogen) as described previously (18 (link)). For tissue immunofluorescence analyses, paraffin-embedded skin sections were incubated with ASMA (Abcam, 1:100, ab5694), p-p65 (Cell Signaling Technology, 1:100, 3033), Fn-EDA (Abcam, 1:100, ab6328), tenascin-C (Abcam, 1:100, ab108930), RP105 (Abcam, 1:100, ab184956), and MD1 (Santa Cruz, 1:50, sc-390613), followed by appropriate secondary antibodies. Nuclei were detected using DAPI. Slides were mounted, and immunofluorescence and immunohistochemistry were evaluated under Nikon A1R laser scanning confocal microscope in a blinded manner. Negative controls stained without primary antibodies were used to confirm immunostaining specificity.

Liver tissue sections were stained for α-SMA, Abcam (ab5694) (Cambridge, UK), SPARC, Cell Signaling Technology (D10F10) (Danvers, USA), COL-1A, Abcam (ab90395) (Cambridge, UK), pan-Cytokeratin, Ventana Medical Systems (AE1/AE3/PCK26) (Tucson, Arizona, USA) and PCNA, Cell Signaling Technology (PC10) (Danvers, USA).
Formalin-fixed and paraffin-embedded tissue sections were deparaffinized in xylene and graded alcohols, hydrated, and washed in phosphate buffered saline (PBS). After an antigen retrieval in a sodium citrate buffer (pH 6) in a microwave oven, the endogenous peroxidase was blocked by 0.3% H₂O₂ for 15 min. Sections were incubated overnight at 4°C with primary antibody (1 : 100) and as a secondary antibody; a horseradish peroxidase (HRP) SuperPicTure Polymer detection kit was used (Invitrogen). As a chromogen substrate for HRP, either DAB or ACE (Dako) was used. A matched isotype control was used as a control for nonspecific background staining, and a routine standard staining showing a normal histology of liver sections from respective groups was performed with hematoxylin/eosin and Masson's Trichrome, respectively. The proliferative index was assessed by counting the number of PCNA-positive cells at ×200 magnification in five randomly chosen fields per slide in three animals, one at 0, one at 3, and one at 6 weeks after PHx.