Alexa 488 and 568 conjugated secondary antibodies

To assess the localization of vWF and α-SMA in liver tissues, cryosections (7 µm thick) were fixed in 100% methanol (Merck) and incubated with blocking solution (Dako) for 1 h at RT followed by anti-vWF (1:200, Abcam) and anti-α-SMA (1:400, Dako) primary antibodies at 4 °C overnight. The sections were incubated with Alexa 488- and 568-conjugated secondary antibodies (1:400; Invitrogen) for 1 h at RT. To evaluate the localization and expression of α-SMA in rat HSCs, T-HSCs treated with WKYMVm were fixed with 100% methanol. The cells were incubated with blocking solution (Dako) at RT for 40 min and a rabbit anti-α-SMA (1:200) antibody at 4 °C overnight. Subsequently, the cells were incubated with an Alexa 568-conjugated secondary antibody (1:400, Invitrogen) at RT for 1 h and counterstained with DAPI. Images were taken with a confocal microscope (LSM 700) and analyzed with ImageJ software. All experiments were performed in triplicate.

F12 and F12 (Coon’s modification) medium, latrunculin B, cycloheximide (Cat. No. C7698-1G) was from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) and newborn calf serum were from Gemini Bio-Products (Woodland, CA, USA). Anti-α-tubulin mouse monoclonal antibodies (DM1A, Cat. No. T9026, lot no. 052M4837), anti-FLAG mouse monoclonal antibodies, were from Sigma-Aldrich. Rabbit polyclonal antibodies against full length human MAL2 (Cat. No. ab75347), and VASP (Cat. No. ab47461) were from AbCam (San Francisco, CA, USA). Rabbit polyclonal antibodies against Tks5 (Fish (M-300)) were from Santa Cruz (Dallas, TX, USA) (Cat. sc-30122). Rabbit polyclonal antibodies against Mena (Cat. No. NBP1-87914) were from NOVUS (Littleton, CO, USA). Rabbit polyclonal antibodies against rat MAL2 were generated and affinity purified as described previously [4 (link)]. Monoclonal antibodies against rat zonula occludens-1 (ZO-1) were generously provided by Dr. Ann Hubbard (Johns Hopkins University School of Medicine, Baltimore, MD, USA) and are now part of our lab stocks. Alexa488 and -568-conjugated secondary antibodies were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA) and HRP-conjugated secondary antibodies were from Sigma-Aldrich.