Anxa3

We collected 13 burn patient skin samples (deep second-degree burn tissue) from 2020 to 2021 from burn patients associated with the Chinese Han population. We received approval from the subject review committee of Foshan First People’s Hospital, and the patients signed an informed consent form and were informed prior to sample collection. After dehydration, formalin-fixed and paraffin-embedded skin tissues were sectioned at 4 μm for IHC. Antigen retrieval was performed by incubating the samples in citrate buffer (pH 6.0) for 15 min. After blocking with a mixture of methanol and 0.75% hydrogen peroxide, sections were incubated overnight with primary antibodies (ANXA3, Abcam, Cambridge, United Kingdom, 1:100; MCEMP1, Abcam 1:150; S100A12, Signalway Antibody, MD, United States, 1:100; TCN1, Invitrogen, MA, United States, 1:50; MMP9, Signalway Antibody, 1:50), followed by incubation with a secondary antibody conjugated with horseradish peroxidase (goat anti-rabbit, 1:500, Cell Signaling Technology, MA, United States). Sections were washed three times with phosphate-buffered saline and incubated with diaminobenzidine. Next, we performed a comprehensive IHC score according to the total degree of staining and the area of positive cells, and the scoring criteria and steps described in our previous article (Zhou et al., 2021 (link)).

Total protein was extracted from cultured cell lines using RIPA lysis buffer (Beyotime, China) and a BCA protein kit (Beyotime, China) was used to assess protein concentrations. Protein samples of cSCC cells were separated on 4–20% SDS-PAGE gels and transferred onto a PVDF membrane (Millipore, USA). After blocking with 5% milk-TBST, membranes were incubated with primary antibody at 4 °C overnight and then probed with HRP-conjugated anti-mouse/rabbit secondary antibodies (1:2000; Cat# ab205719, Cat# ab6721 Abcam, USA) at RT for 1 h. Protein bands were detected using the SuperSignal West Pico PLUS Kit (Thermo Scientific, USA). The primary antibodies included 4EBP1 (1:2000; Cat#ab32024), RAP2B (1:1000; Cat#ab101369), ANXA3 (1:1000; Cat#ab127924), and GAPDH (1:10,000; Cat#ab8245), all of which were purchased from Abcam (Cambridge, USA).