Hrp conjugated anti mouse igg secondary antibody

To investigate the endothelial differentiation of encapsulated cells after 7 days being enclosed by the alginate-gelatin hydrogel, we measure the protein content of VE-cadherin by ELISA. In brief, 100 μL protein was transferred onto each well of polystyrene 96-well plates (SPL) and maintained at 4°C overnight. Next, wells were blocked with 1% FBS for 1 h. Thereafter, we added 100 μL mouse anti-human VE-cadherin antibody (dilution 1: 100; Abcam) and kept for 1 h. After twice washing with PBS, we added HRP conjugated anti-mouse IgG secondary antibody (1: 1000; Abcam), incubated for 30 min and washed three times with PBS. 3, 3’, 5, 5’-Tetramethylbenzidine was used as chromogenic substrate and the reaction stopped by using 5% H₂SO_4. Finally, the absorbance was read at 450 nm by using microplate readers (BioTek).

SARS-CoV-2 nucleocapsid-specific antibodies were detected by ELISA as in our previously reported method [26 (link),27 (link)]. In brief, 96-well EIA plates (Corning Inc, Corning, NY, USA) were coated with 100 ng per well of SARS-CoV-2 N protein antigens (Abclonal, Wuhan, China) in PBS overnight at 4 °C. Plates were washed with PBST three times and then blocked with 5% skimmed milk in PBST for 1 h at 37 °C. Mouse serum was diluted at 1:25, and then added to the washed plates and incubated for 2 h at 37 °C. After washing, plates were incubated with a 1:5000 dilution of HRP conjugated anti-mouse IgG secondary antibody (Abcam, Cambridge, UK), and HRP conjugated anti-mouse IgG1 secondary antibody (Abcam), HRP conjugated anti-mouse IgG2a secondary antibody (Proteintech, Wuhan, China), and HRP conjugated anti-mouse IgG2c secondary antibody (Abcam, Cambridge, UK), respectively, for 1 h at 37 °C. The color reaction was substrated with 3,3′,5,5′-Tetramethylbenzidine (TMB) and stopped with 1 M H₂SO₄. The value was read at a 450 nm wavelength using a Synergy HT Multi-Mode Plate Reader (BioTek, Winooski, VT, USA).

Immunoblotting was performed as previously described (23 (link)). The antibodies used in this study were as follows: mouse anti-GAPDH antibody (1:20,000; Santa Cruz; sc-32233), mouse anti-α-SMA antibody (1:10,000; Thermo; MS-113-P), rabbit anti-SM22α antibody (1:4000; Abcam; ab14106), rabbit anti-Col1a1 antibody (1:4,000, CST; 72,026), rabbit anti-β-actin antibody (1:4000; Proteintech; 20536-1-AP), rabbit anti-Mkl1 antibody (1:4000; Proteintech; 21166-1-AP), rabbit anti-lamin A/C antibody (1:4000; Proteintech; 10298-1-AP), rabbit anti-SOX9 antibody (1:4000; abcam; ab185230), horseradish peroxidase (HRP)-conjugated anti-FLAG primary antibody (1:10,000; Sigma Aldrich; A8592), horseradish peroxidase (HRP)- conjugated anti-HA primary antibody (1:5000; Roche; 12013819001), HRP-conjugated anti-mouse IgG secondary antibody (1:10,000; Abcam; ab6789), HRP-conjugated anti-rabbit IgG secondary antibody (1:10,000; Abcam; ab6721), HRP-conjugated anti-rabbit IgG secondary antibody (1:10,000; Santa Cruz; sc-2004), and HRP-conjugated anti-rabbit IgG secondary antibody (1:2000; CST; 7074).