Ab8592

Histological slides were prepared with hematoxylin-eosin (HE) stain and were used to mark areas of interest for performing tissue sample array arrangement or Tissue Microarray (TMA). Next, two 4 µm-thick paraffin-embedded sections of the TMA blocks were transferred to electrically charged Star Frost™ (Braunschweig, Germany) slides and incubated with an anti-Desmine (ab8592; Abcam, Cambridge, UK), anti-connexin 43 (ab217676; Abcam, Cambridge, UK) and anti-factor VIII (275376; Abcam, Cambridge, UK) overnight in a humidified chamber at a temperature between 2–8 °C [28 (link)].
Immunoreactivity was developed by adding DAB chromogen-substrate solution (Spring) to the slides. Harris hematoxylin was used for counterstaining. Positive and negative controls were performed in parallel with all reactions. The slides were scanned using the Axio Scan.Z1 scanner (Carl Zeiss, Jena, Germany). The generated files were fragmented into single images and approximately 25 images were selected for analysis. The areas of immunopositive markings for the antiprotein antibodies were quantified using Image-Pro Plus version 4.5 software (Media Cybernetics, Rockville, MD, USA). The immunopositive objects were selected using a “mask” to standardize and automate the process. Numerical data of the immunopositive marking area were generated and subsequently exported to an Excel spreadsheet.

Protein analyses of whole liver tissue lysates were performed using the semidry Western blot technique according to standard protocols. Antibodies against α‐SMA (M0851; Dako/Agilent), desmin (ab8592; Abcam), GFAP (MAB360; Merck/Millipore), fibronectin (610078; Becton Dickinson), ITGA5 (ab150361; Abcam), ITGB1 (ab52971; Abcam), HGF (LS‐C486534; Lifespan Biosciences), and γ‐tubulin (T5326; Sigma‐Aldrich) were used. The detection was carried out using horseradish peroxidase (HRP)‐coupled goat‐anti‐mouse or goat‐anti‐rabbit antibodies (Merck/Millipore) and Western Bright Quantum or Sirius HRP substrates (Advansta). Visualization and documentation was performed with ChemiDoc Imaging System (BioRad). The intensities of Western blot protein bands were analyzed using the multiplex band analysis tool of the AlphaView software (Proteinsimple) and normalized to the intensity of γ‐tubulin bands.

Cells were fixed with 4% paraformaldehyde (PFA) for 5 min at room temperature, then permeabilized and blocked with 0.5% Triton X-100 and 10% goat serum for 20 minutes at room temperature. Cells were incubated in primary antibody for 1 h at room temperature at a 1:30 ratio for anti-desmin (ab8592, Abcam), anti-dystrophin (NCL-DYS1, Novocastra), and anti-myosin heavy chain (NCL-MHCf, Novocastra). Cells were incubated with 1:500 anti-rabbit or mouse IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 594 (Invitrogen). SlowFade Gold Antifade Mountant with DAPI (Invitrogen) was added and cells were stored at 4°C.