Rabbit monoclonal anti α sma

The specific operation process was the same as before (18 (link)). Isolated PSCs from different sources which were passaged 2-3 times were inoculated into a six-well culture plate equipped with a cover slip, cultured under standard conditions for 48 hours, and then incubated with primary antibody (rabbit monoclonal anti-α-SMA, 1:100, Abcam, USA). The cells were detected and imaged with a fluorescence microscope, and the purity of PSCs was analysed.

IF was used to character the classic markers of CAFs. Cells were digested and implanted on cover glass beforehand. After washed three times using PBS, 10% formaldehyde solution fixed. The cover glass was incubated with rabbit monoclonal anti-α-SMA (1:500, Abcam, UK, Cat: ab7817) and rabbit monoclonal anti-FAP (1:500, Abcam, UK, Cat: ab314456) overnight at 4℃. Then the secondary antibody was incubated with the samples for 45 min at 37℃ in dark conditions. DAPI was used to stain the nucleus for 10 min.

Tissue multi-immunofluorescence was done to validate the expression of EPCAM, α-SMA and CD146 in endometrial cancer. The tissue sections were deparaffinized, hydrated and rehydrated according to the standard protocols. Antigen retrieval was performed by high-temperature heating (EDTA, pH9.0) for ninety seconds. Endogenous peroxidase and non-specific binding sites were blocked by 10% serum consistent with secondary antibodies for thirty minutes in 37℃. The sections were incubated with rabbit monoclonal anti-EPCAM (1:500, Abcam, UK, Cat: ab223582) overnight at 4℃ and the secondary antibody was incubated with the samples for 45 min at 37℃. Tyramide signal amplification (TSA) stain was carried out as standard. Then the sections were repeated the above steps while different antibodies (Rabbit monoclonal anti-α-SMA, 1:500, Abcam, UK, Cat: ab7817, Rabbit monoclonal anti-CD146, 1:250, Abcam, UK, Cat: ab75769) were used. DAPI was used to stain the nucleus in dark conditions.

Proteins of mouse retina tissue were extracted with a protein extraction solution assay kit (RAPI, Sigma). Nanodrop (Thermo) was used to measure protein concentration. Equal amounts of proteins were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) resolving gel and 5% stacking gel and transferred onto polyvinylidene fluoride (PVDF) membranes. PVDF membranes were blocked with skim milk (BD) in Tris-buffered saline (TBS) for 1 h at room temperature. Following this, they were incubated with primary antibodies at 4 °C overnight (rabbit monoclonal anti-ZO-1, 1:500, Abcam; mouse monoclonal anti-RPE65, 1:500 Abcam; mouse polyclonal anti-Nestin, 1:500 Santa Cruz; rabbit monoclonal anti-α-SMA, 1:1000, Abcam; mouse polyclonal anti-CD68, 1:1000, Proteintech; rabbit anti-Bax, 1:1000, Abcam; rabbit monoclonal anti-GAPDH, 1:1000, Abcam). This was followed by PVDF membranes being washed with TBS-T (Tween 1:1000 dilution in TBS buffer) three times followed by incubation with secondary antibodies (HRP-goat anti-rabbit IgG, 1:2000, Bioss; HRP-goat anti-mouse IgG, 1:2000, Bioss) for 1 h at room temperature. After incubation, PVDF membranes were washed three times with PBS. Protein signals were detected with SuperSignal™ West Femto Maximum sensitivity substrate (Thermo Fisher Scientific) and imaged using the chemiluminescence system (Bio-Rad).