Anti calponin

IHC staining was carried out as previously described [15 (link)]. In brief, tissue Sections (4 μm) were dewaxed in xylene twice for 2 min each and then rehydrated in a graded series of ethanol (100–70%). Antigen retrieval was performed by boiling sections for 15 min in sodium citrate buffer (10 mM citrate acid, 10 mM sodium citrate, pH 6.0). Then, 5% normal donkey serum was used to block nonspecific antigens. IHC was performed using the Dako EnvisionTM method for antibody incubation and then developed by using the DAB peroxidase substrate kit (Beyotime, P0202). IHC-stained sections were imaged by a Leica LF200 microscope.
Antibodies for IHC included anti-S100 (Dako Z0311, 1:200), anti-Calponin (Dako M3556, 1:100), anti-SMA (Dako M0851, 1:100), anti-CK7 (Dako M7018, 1:50) and anti-CK19 (Dako M0888, 1:100), anti-FZD2 (Bioworld BS3163, 1:50), which were purchased from commercial sources.

Cells seeded on microscope coverslips were fixed with 4% formaldehyde for 20–30 min, permeabilized with 0.4% Triton X-100 in PBS for 10–20 min, and blocked with 10% donkey serum in PBS for 1 h at room temperature. Cells were then incubated with primary antibody (diluted with 1% donkey serum in PBS) overnight at 4 °C and fluorescence-labeled secondary antibody (Invitrogen; 1:500 diluted with 1% donkey serum in PBS) at room temperature for 1 h the next day. Hoechst 33342 (Invitrogen; 1:1,000) was used to stain nuclear DNA.
Primary antibodies for immunofluorescence include anti-NANOG (Abcam, ab21624; 1:100), anti-SOX2 (Santa Cruz, sc-17320; 1:200), anti-OCT4 (Santa Cruz, sc-365509; 1:200), anti-TUJ1 (Sigma, T2200; 1:500), anti-α-SMA (Sigma, A5228; 1:200), anti-FOXA2 (CST, 8186S; 1:200), anti-Vimentin (Abcam, ab8978; 1:250), anti-α-Tubulin (Sigma, T5168; 1:500), anti-Vinculin (Sigma, V9131; 1:100), anti-ZOI (Abcam, ab96587; 1:200), anti-ClaudinV (Abcam, ab15106; 1:200), anti-SM22 (Abcam, ab14106; 1:200), anti-Calponin (Dako, M3556; 1:200), anti-vWF (Dako, A0082; 1:200), anti-eNOS (BD, 610296; 1:100), anti-VE-cadherin (CD144) (CST, 2158S; 1:100), anti-human CD31-FITC (BD biosciences, 557508, 1:100), anti-Ki67 (Vector Laboratories, ZA0731; 1:500), Phalloidin (F-actin) (Invitrogen, A22287; 1:50), anti-NF-κB P65 (RelA) (CST, 8242S; 1:200).