Western blot analyses and immunostaining were performed with GAPDH (MAB374, EMD Millipore), phospho-MLC2v (gift from Dr. N. Epstein, NIH) (Davis et al., 2001 (link)), MLC2 (F109/3E1. ALX-BC-1150-S-L005, Enzo Life Science), and cMLCK (Chan et al., 2008 (link)) antibodies. The rabbit affinity-purified cMLCK antibody production has been described previously (Chan et al., 2008 (link)). Briefly, the antigen was purified from GST-cMLCK(aa 28-463) following cleavage of GST-tag using thrombin and SDS-PAGE separation followed by electro-elution. Rabbits were injected with the antigen and adjuvants 4 times, and the serum from these animals were affinity-purified using GST-cMLCK(aa 28-463) covalently-coupled beads. 1:4000-diluted antibody was used for Western blotting.
Histological sections (5 μm) were analyzed by hemotoxylin and eosin staining, as well as for fibrosis after Picrosirius red staining, which was performed by heating the tissue sections at 60⋅C for 45 min before deparaffinization and then immersing them in a combination of 0.1% direct red 80 and 0.1% fast green FCF in 1.2% picric acid for 1 h. TUNEL staining was performed using the In Situ Cell Death Detection Kit (Roche 11684795910).
Histological sections (5 μm) were analyzed by hemotoxylin and eosin staining, as well as for fibrosis after Picrosirius red staining, which was performed by heating the tissue sections at 60⋅C for 45 min before deparaffinization and then immersing them in a combination of 0.1% direct red 80 and 0.1% fast green FCF in 1.2% picric acid for 1 h. TUNEL staining was performed using the In Situ Cell Death Detection Kit (Roche 11684795910).