Clone d5 16 b4

Antibodies (all from Dako North America, Carpintería, CA, USA) against cytokeratins (CKs) AE1/AE3 (clones AE1/AE3; dilution 1:50) and CK 5/6 (clone D5/16 B4; dilution 1:50) and against chromogranin (clone DAK-A3; dilution 1:100), synaptophysin (clone DAK-SYNAP; dilution 1:200), CD56 (clone 123C3; dilution 1:50), CD99 (clone 12E7; dilution 1:1000), neuronal specific enolase (clone BBS/NC/VI-H14; dilution 1:200), Ki67 (clone MIB-1; dilution 1:100) and PD-L1 (clone 22C3; dilution 1:50) were used for the characterization of each of the tumors through immunohistochemistry studies. Expression of PD-L1 was considered positive when there was ≥1% of tumor cells with membranous staining [10 (link)].

Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded tissue TMA sections. Briefly, 3 μm-thick TMA tissue sections were placed onto adhesive slides and treated with the PT link (DAKO) solution. Immunohistochemical staining was performed using the following primary antibodies: anti-Fatty Acid Synthase polyclonal antibody (1:100, Enzo Life Sciences), anti-EGFR monoclonal antibody (1:100, clone D38B1, CellSignaling), anti-Cytokeratin 5/6 monoclonal antibody (1:50/100, Clone D5/16 B4, DAKO) anti Monoclonal Mouse Anti-vimentin (1:100/200, Clone Vim 3B4, DAKO). Sections were washed with PBS and sequentially incubated at room temperature for 45 minutes with antirabbit or antimouse IgG. Immunodetection was performed with the kit EnVision™ (DAKO, Glostrup, Denmark) using the AutostainerPlus Link (DAKO).

Skin samples were fixed in 4% neutral buffered paraformaldehyde solution at room temperature for at least 24 h, embedded in paraffin blocks (Shandon Cytoblock, Thermo Scientific, USA), cut into 4 μm sections and stained with haematoxylin, eosin and saffron (H&E). Immunohistochemical procedures were carried out using an automated immunohistochemical apparatus according to the manufacturer’s instructions (Bond-Max slide stainer, Leica Microsystems).
Briefly, after dewaxing, paraffin sections were rehydrated, and antigen retrieval was performed in an Antigen Retrieval Buffer (Leica Biosystems) at pH 9. Sections were incubated for 30 min with primary antibodies, rinsed, and then incubated with a biotinylated secondary antibody. Sections were rinsed and the reaction was developed according to the manufacturer’s guidelines (streptavidin-peroxidase with an automated BOND, Leica Microsystems). Primary antibodies were used at the indicated dilutions: GATA6 (1:750, D61E4 clone, Cell Signalling 5851), Ki67 (1:100, Clone MIB-1, Dako M7240) and KRT5/6 (1:100, Clone D5/16 B4, Dako M7237). Biotinylated secondary antibodies (Vector Laboratories) were used at 1:400 dilution. Images were acquired with a Pannoramic 250 Flash slide scanner (3DHistech) and visualised with QuPath version 0.1.2 (https://qupath.github.io).