Nb300 978ss

Human intestinal fibroblasts were fixed in 3.7% formaldehyde, washed, and subjected to immunofluorescent staining. Next, 1% BSA in phosphate buffered saline (PBS) was used to block unspecific binding sites. Polyclonal goat antihuman ACTA2 (NB300-978SS, Novus Biologicals, diluted 1:400) was applied. Fluorescent labeling was performed with Alexa Fluor 647 donkey antigoat (#A32849; Invitrogen; diluted 1:1'000). Nuclei were visualized with diamidino phenylindole (D1306, DAPI; Invitrogen; final concentration 3 μM). The sections were mounted with fluorescent mounting medium (S302380, DAKO, Switzerland) and analyzed by confocal laser scanning microscopy (SP5, Leica, Switzerland). The colors were assigned with the help of the software LAS AF lite.

Samples were lysed in M-PER protein extraction buffer (Thermo Fisher Scientific). Proteins were separated on 12% polyacrylamide gels with Tris/SDS running buffer and transferred onto nitrocellulose (Invitrogen). Membranes were blocked with 5% milk, 3% bovine serum albumin (BSA) and 0.1% Tween 20 and incubated with polyclonal rabbit antimouse COL1A1 (91144S; Cell Signaling; diluted 1:1'000), monoclonal rabbit antimouse VIM (5741S, Cell Signaling; diluted 1:1'000), polyclonal rabbit antimouse TGFβ1 (3711S, Cell Signaling; diluted 1:1'000), polyclonal goat antimouse. IL-1β (AF-401-NA, Bio-techne, diluted 1:1'000) and polyclonal goat antihuman ACTA2 (NB300-978SS, Novus Biologicals, diluted 1:1'000). Horseradish peroxidase-conjugated secondary antibodies were donkey antigoat (#sc-2020; Santa Cruz; diluted 1:3'000) and goat antirabbit (#sc-2004; Santa Cruz; diluted 1:3'000). Additionally, β-Actin was used as a loading control.