Foxp3 monoclonal antibody

Assessment of H&E stained sections from lesional skin of different studied groups was done under light microscopy (Olympus, CXR-23). Full epidermal thickness was measured in microns by recording the distance between the top of the epidermis and the bottom of the rete ridges. At least four different measurements were recorded in each sample and then average was calculated.
Immunostaining by both FOXP3 monoclonal antibody (Thermo Fisher Scientific, Cat # 14-5773-82) with a dilution of 1:200 and RORγ polyclonal antibody (Bioss, Cat # bs-6217R) with a dilution of 1:200 was done using Envision detection system (Dako autostainer Link48) applying DAB as chromogen and hematoxylin as a counterstain. Positive lymphocytes within dermal inflammation were counted per one high power field (HPF) using Image J software. Then the mean number/ 5 HPFs was calculated (Fondi et al. 2009 (link)).

After anaesthesia with sodium pentobarbital (40 mg/kg, i.p.), blood was extracted from the heart and placed in tubes containing the anticoagulant ethylenediaminetetraacetic acid (EDTA), and plasma was separated via centrifugation.
The levels of interleukin-6 (IL-6 ELISA Kit, Cusabio Biotech, CSB-E04640r, Wuhan, China), interleukin-10 (IL-10 ELISA Kit, Cusabio Biotech, CSB-E04595r, Wuhan, China), and interleukin-17A (IL-17A ELISA Kit, Cusabio Biotech, CSB-E07451r, Wuhan, China) were measured using ELISAs.
Th17 cells were labeled with CD4-FITC and IL-17A-PE; Tregs were labeled with CD4-FITC, CD25-PerCP, and FoxP3-PE. A rat peripheral blood lymphocyte isolation kit (Solarbio, P8630, Beijing, China) was used to separate lymphocytes according to the manufacturer's protocol. Then, 6 μL cocktail stimulation solution and 4 μL GolgiStop protein inhibitor were added to the lymphocytes, and the mixture was gently mixed and incubated in the dark at 37°C for 5 h. The cell surface was stained with IL-17A monoclonal antibody (Thermo Fisher Scientific, 45-7177-82, Waltham, MA, USA) or Foxp3 monoclonal antibody (Thermo Fisher Scientific, 56-5773-82, Waltham, MA, USA), and the cells were fixed and permeabilized. Intracellular staining was performed. Th17 cells and Tregs were detected via flow cytometry.

Ovalbumin, PMA, and 1A,245-dihydroxyvitamin D3 were obtained from Sigma-Aldrich (St. Louis, MO, USA). Aluminum hydroxide gel, FOXP3 monoclonal antibody, and IL-17 monoclonal antibody were purchased from Thermo Fisher Scientific (Shanghai, China). CD4 monoclonal antibody and CD 25 monoclonal antibody were purchased from Invitrogen (Carlsbad, CA, USA).