Hrp labelled goat anti mouse igg

Immunohistochemical (IHC) analyses were performed to study p62 protein expression in the 26 tissues. The tissues were embedded in paraffin and then cut into 6-µm-thick sections. These sections were dewaxed in xylene and then rehydrated in an ethanol series. Pressure cooking in citrate buffer (pH = 6) (Beyotime, China) for 5 min was applied to for antigen retrieval. Next, blocking of endogenous peroxidase was performed in 0.3% H₂O₂ for 10 min. Incubation with the p62 primary antibody (dilution 1:1000, AB56416, Abcam, USA) occurred overnight at 4 °C. Detection was performed with HRP-labelled goat anti-mouse IgG (dilution 1:2000, A0216, Beyotime, China) for 30 min, followed by incubation with the DAB chromogen with a DAB horseradish peroxidase chromogen kit (Beyotime, China). Sections were counterstained in haematoxylin (Beyotime, China) for 10 s and dehydrated in an ethanol series.

For western blotting, MuSCs were digested using 0.25% trypsin and lysed using RIPA buffer, and total protein concentration was measured using a BCA kit (Beyotime, P0010S). Proteins were separated by SDS-PAGE; subsequently, the separated proteins were blocked using 5% fat-free milk for 2 h at room temperature. After incubating the proteins with primary antibodies at 4 °C, secondary antibodies were added, and the protein complex was visualized. The following antibodies were used: HEB (A-6) (Santa, 1:500), β-tubulin (ABclonal, 1:1000), HRP-labelled goat anti-rabbit IgG (Beyotime, A0208) and HRP-labelled goat anti-mouse IgG (Beyotime, A0216). For immunofluorescence staining, cells or frozen sections were fixed in 4% PFA and blocked with goat serum at room temperature for 1 h. Primary antibodies were added and incubated at 4 °C overnight. The following antibodies were used: Pax7 (DSHB, 1:100), myosin heavy chain (MHC; DSHB, 1:400), embryonic MHC (eMHC; DSHB, 1:500), laminin (Sigma, 1:500) and TCF12 (Abclonal, 1:300). Secondary antibodies were purchased from Invitrogen and were used according to the manufacturer’s instructions. For Pax7 staining, antigen retrieval was performed using 10 mM trisodium citrate dihydrate, which was incubated for 30 min in a 70 °C water bath. All staining images were captured using a fluorescence microscope.