Sc 100289

Paraffin blocks were cut into 1 µm-thick sections and fixed onto a poly-L-lysine treated glass slide and then used for histological and immunohistochemical analyses. All sections were stained with H&E following manufacturer’s protocol to achieve correct orientation of the biopsy. Immunohistochemical staining was performed using Bond Polymer refine Detection kit on a BOND-MAX Automated IHC/ISH Stainer (Leica Biosystems) following the manufacturer’s protocol. The following antibodies were used: mouse monoclonal anti-human CD19 (NCL-L-CD19-163, Leica Biosystems), rat anti-human CD3 (MCA1477,Bio-Rad), mouse monoclonal anti-human CD4 (NCL-L-CD4-368, Leica Biosystems), mouse monoclonal anti-human CD8 (NCL-L-CD8-4B11, Leica Biosystems), mouse monoclonal anti-human Claudin-1 (ab56417, Abcam), mouse monoclonal anti-human E-Cadherin (36B5) (PA0387, Leica Biosystems), mouse monoclonal anti-human FoxP3 (ab22510, Abcam), mouse monoclonal anti-human γδ-TCR (sc-100289, Santa Cruz Biotechnology), mouse monoclonal anti-human Langerin (ab49730, Abcam), rabbit recombinant monoclonal [EPR20992] to Occludin (ab216327, Abcam), and rabbit polyclonal to Neutrophil Elastase (ab68672, Abcam). Positive and negative controls were included for each experiment.

The cohort was classified based upon morphology of cutaneous lesions, characteristics of the atypical infiltrate on histology, and the presence or absence of cytotoxic cell markers (TIA-1, granzyme B, perforin). Clinical information was obtained from the electronic medical record of each patient when available and is summarized in Supplementary Table 1. Metastatic spread was determined on the basis of positron emission tomography-computed tomography (PET–CT) or computed tomography (CT) imaging highly suspicious for metastatic involvement. One-third of these cases had metastasis proven by biopsy and/or cytology. Antibodies used to determine γδ TCR expression included γ3.20 antibody clone (TCR1153, ThermoFisher Scientific, IL) or H-41 antibody clone (SC-100289, Santa Cruz Biotechnology, TX)^{60 ,61 (link)}. Major pathological criteria that were assessed were depth of infiltrate involvement, ulceration, keratinocyte necrosis, and vasculitis. Samples were divided in groups based on depth of skin involvement: (1) epidermal (2) epidermal/dermal, and (3) panniculitic. There was insufficient clinical annotation to distinguish between epidermal vs. epidermal/dermal involvement in five samples. Biopsy specimens were subject to review by expert pathologists (J.G., A.L., and/or A.G.).

We used the following primary antibodies for single-color IHC. Hypoxia-inducible factor-1 alpha (HIF1α, mouse monoclonal, clone 54, 610959, BD Biosciences, San Jose, CA; 1:100, 3-h incubation), CD31 (mouse monoclonal, clone JC70A, M0823, DAKO, Carpinteria, CA; 1:50, 1-h incubation), basic fibroblast growth factor (bFGF, mouse monoclonal, clone bFM-2, 05-118, Upstate/Millipore, Billerica, MA; 1:200, 1-h incubation), VEGF-A (rabbit polyclonal, sc-152, Santa Cruz Biotechnology, Santa Cruz, CA; 1:200, 1-h incubation), T-cell receptor delta chain (TCR δ-chain; mouse monoclonal, sc-100289, Santa Cruz; 1:400, overnight incubation).
We used the following primary antibodies for two-color immunofluorescence (IF). Angiopoietin-2 (Ang2, mouse monoclonal, clone MM0020-1F29, sc-101441, Santa Cruz Biotechnology; 1:40, 4-h incubation), alpha-smooth muscle actin (αSMA, mouse monoclonal, clone alpha sm-1, PA0943, Leica Microsystems, Inc., Buffalo Grove, IL; ready-to-use, 15-min incubation), CD31 (mouse monoclonal, clone JC70A, M0823, DAKO; 1:40, 3-h incubation).