Ab22378

All reagents were purchased from Sigma (St. Louis, MO) unless indicated otherwise. Dyes and secondary antibodies were obtained from Thermo-Fisher (Waltham, MA). RNAscope 2.5 HD Detection of s-sense COVID-19 for RNA detection was used (Hayward, CA). Antibodies for macrophages (Iba-1, Ab5076), lymphocytes (CD3, ab11089), endothelial cells (Von Willebrand factor, ab194405), epithelial cells (EpCam, ab7504), myeloperoxidase (MPO, ab25989), CD8 (CD8, ab22378), CD20 (B cells, ab9475), and smooth muscle actin (SMA, ab21027) were obtained from Abcam, MA. Vimentin (sc-52721) from Santa Cruz (Santa Cruz, CA) and the antibody for SARS protein M (APO90991su-n) were obtained from Origene (Rockville, MD). All experiments were performed under the University of Texas Medical Branch (UTMB) and the NIH regulations.

Antidifferentiation cluster CD3, CD8, and interferon-γ (IFN-γ) antibodies were used to detect CD3⁺CD8⁺IFN-γ⁺ cells by flow cytometry. The cells were incubated for 30 min at 4°C with rat monoclonal antibodies: fluorescein isothiocyanate- (FITC-) conjugated antibodies (Abcam) to CD8 (ab22378, 1: 500) and IFN-γ (ab175878, 1: 100) and PE-conjugated antibody to CD3 (ab135372, 1: 200, Abcam). Following the fixing with 1% paraformaldehyde in PBS, cells were analyzed by a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). Allotype-matched antibodies were adopted to control nonspecific staining subtracted from the specific staining results. At least 10,000 cells per sample were analyzed with the WinMDI 2.8 software (J. Trotter, Scripps Research Institute, La Jolla, CA).

After resection, tumors were digested to obtain a single cell suspension. Tumor infiltrating lymphocytes (TILs) were isolated from 2×10⁶ single-cell suspension derived from the homogenated tumors, using the CD8 (TIL) MicroBeads (mouse, 130-116-478, Miltenyi Biotec., Bergisch Gladbach, Germany) and the CD4 (TIL) MicroBeads (mouse, 130-116-475, Miltenyi Biotec.), in order to obtained a purified population of TILs that represent a small percentage of cells contained in the tumor mass, ready for the quantification by flow cytometry. TIL quantification was performed using a Guava EasyCyte flow cytometer (Millipore, Bedford, MA), equipped with the InCyte software (Millipore). For immunohistochemical analyses, tumors sections were stained for Ki67 (AB9260, Millipore), CD4 (ab183685, Abcam, Cambridge, UK), CD8 (ab22378, Abcam) antibodies, followed by a secondary HRP-labelled antibody (Dako, Glostrup, Denmark). For the detection of intratumor apoptosis, tumor sections were treated with the In Situ Cell Death Detection Kit (11684795910, Sigma Chemicals Co.) based on TUNEL staining. Sections were examined with a Leica DC100 microscope (Leica Microsystems GmbH Wetzlar, Germany). Immunohistochemical imaging quantification was performed with ImageJ software (https://imagej.nih.gov/).

For Th17 cell detection, the above collected splenocytes or spinal cord cells were prepared into single cell suspension according to flow cytometry, about 10⁶ cells per tube. CD4 antibody (0.25 μL) labeled with fluorescence was put into each tube and incubated for 30 min without exposure to light. The fluorescent labeled IL-17A antibody (1:250, ab79056, Abcam) was added to the fixed membrane breaker and buffer after proper treatment, and incubated at room temperature for 60 min without exposure to light. Next, cells were resuspended with fixed membrane breaker buffer, and centrifuged to discard the supernatant. Finally, cells were resuspended with 2 mL flow staining solution and loaded on the flow cytometer.
For Treg cell and T cell detection, the collected splenocytes or spinal cord cells were prepared into single cell suspension according to flow cytometry, about 10⁶ cells per tube. Treg cells were detected via CD4 (1:250, ab59474, Abcam), CD25 (1:250, ab210330, Abcam) and Foxp3 (1:250, ab210232, Abcam) antibodies, and T cells were detected by CD4 and CD8 (1:250, ab22378, Abcam) antibodies according to Th17 detection procedures.

Lesional skin biopsy specimens from AD patients or OXA-induced mouse tissue specimens were fixed with a 12% formaldehyde solution and embedded in paraffin. For immunofluorescence staining, cells or skin biopsy specimens were incubated with the corresponding primary antibodies at 4°C overnight as follows: anti-K17 (1:1000; ab53707; Abcam), anti-CCL20 (1:300; ab9829; Abcam), anti-CD3 (1:500; ab16669; Abcam), anti-CD4 (1:500; ab183685; Abcam), anti-CD8 (1:500; ab22378; Abcam), anti-STAT3 (1:500; #124H6; Cell Signaling Technology) and anti-p-STAT3 (1:100; #9145; Cell Signaling Technology). After three washes with PBS, Cy3- or fluorescein isothiocyanate-conjugated secondary antibodies (1:200; BioLegend) were added, and Hoechst 33258 (Solarbio Technology, Beijing, China) was applied to label the nuclei. Samples were detected by a confocal microscope (LSM880; Carl Zeiss). For IHC staining, samples were incubated with 0.3% H₂O₂ for 10 min prior to staining with the corresponding primary antibodies: anti-K17 (1:1000; ab53707; Abcam) and anti-CCL20 (1:300; ab9829; Abcam) at 4°C overnight. Sections were subsequently incubated with an HRP-labeled goat anti-mouse/rabbit antibody (CoWin Biosciences) for 1 h at room temperature. 3,3’-Diaminobenzidine (Gene Tech, Shanghai, China) was used to detect biotinylated antibodies.