Quickblock blocking buffer for immunostaining

ELEVL2 (Rabbit pAb (A5918, ABclonal, Wuhan, China)) and CCNB2 (Cyclin B2 Rabbit mAb (A7956, ABclonal, Wuhan, China)) were subjected to immunofluorescence staining experiments. Mouse and pig samples were collected and washed three times with PBS, fixed in 4% paraformaldehyde, embedded in paraffin, and sliced into 5 μm sections. These sections were then held at 6 5 °C for 1 h, deparaffinized, and rehydrated. The sections were finally incubated in citrate antigen retrieval solution (pH 6.0) at 96 °C for 10 min. As the sections gradually cooled, they were blocked in QuickBlock™ Blocking Buffer for Immunostaining (Beyotime, Nantong, China) for 10 min and incubated with primary antibodies at 4 °C overnight. Then, the sections were incubated with secondary antibodies for 30 min at 37 °C, washed with PBST after incubation with antibodies, and incubated with Antifade Mounting Medium containing DAPI (Beyotime, Nantong, China).

Rat footpads were dissected in cold (4°C) 1× phosphate buffered saline (PBS) and fixed in 4% paraformaldehyde solution for 24 h at room temperature (24°C). Paraffin-embedded tissues were sectioned, deparaffinized, and hydrated. The HE staining kit (Beyotime, China) was used for the staining. Briefly, the sections (5-μm-thick) were stained with hematoxylin for 2 min and then immersed in the acidic liquid alcohol for 30 s. The images were obtained using an inverted microscope after staining with eosin for 2 min and dehydrated with ethanol (95, 100%). The samples were blocked with QuickBlock™ Blocking Buffer for Immunostaining (Beyotime) for 10 min, labelled with rabbit anti-Foxa1 antibody (Abcam, 1:400 dilution, UK), mouse monoclonal anti-NKA α2 (1:200 dilution, Santa Cruz, USA) for 8 h at 4°C, and then incubated with Alexa Fluor 546-labeled anti-rabbit IgG (H+L) secondary antibodies (1:500 dilution, Abcam) for 2 h in the dark. For counterstaining, the sections were incubated with 4′,6-diamidino-2-phenylindole (DAPI, Beyotime) in the dark for 1 min at room temperature. Finally, the sections were sealed with an anti-fluorescence quencher. The results were obtained using a Leica DMI8 fluorescence microscope (Leica, Germany).