Hematoxylin dye

Lecithin was purchased from Lipoid GmbH (Ludwigshafen, Germany). All-trans retinoic acid (TRA), fluorescein isothiocyanate (FITC) were purchased from SinopharmDalian Meilun Biotechnology Co., Ltd (Dalian, China). Betamethasone was purchased from Tianjin Tianyao Pharmaceutical Co., Ltd (Tianjing, China). Fetal bovine serum (FBS) was purchased from Hangzhou Tianhang Biotechnology Co., Ltd. (Hangzhou, China). Dulbecco’s minimum essential medium (DMEM), Trypsin-0.02% EDTA was purchased from Geno Biomedical Technology Co., Ltd. (Zhejiang, China). 5% Imiquimod cream was purchased from Hubei Keyi Pharmaceutical Co., Ltd (Hubei, China). ELISA kits (Mouse TNF-α, IL-6) were purchased from Jiangsu Meimian Industrial co., Ltd (Jiangsu, China). Primary antibody (CD3), secondary antibody Hrp-goat anti-rabbit, hematoxylin dye, and eosin stain were purchased from Servicebio co., Ltd (Hubei, China). Bull Serum Albumin (BSA) was purchased from Solarbio co., Ltd (Beijing, China). Other reagents or solvents were chromatographic or analytical pure grade.

After deparaffinization and rehydration, the paraffin sections were stained with a mixture of 2% potassium ferrohydride (Alladin, P112418) and 2% hydrochloric acid (Sinopharm Chemical Reagent, 10 011 008) in equal proportions for 30 min. Then, the sections were washed twice with distilled water and stained with DAB (TCI. D0078) for ≈5 min and washed again. Hematoxylin dye (Servicebio, G1004) staining was performed for 1 min to stain the nuclei, and differentiation was performed with a hydrochloric acid aqueous solution, followed by washing with tap water, immersion in aqueous ammonia solution, and washing with tap water again. Finally, the sections were dehydrated three times with ethanol for 5 min and sealed with neutral resin. Three sections were viewed and photographed under a microscope.