Shandon cytoblock

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).

We retrospectively reviewed archival cell blocks (CBs) with the diagnosis of DLBCL with enough available material diagnosed from November 2016 to January 2019 at Università della Campania Luigi Vanvitelli Hospital. The availability to select representative samples was considered when the sample contained more than 100 cells for the diagnosis and also in the reevaluation on H/E stained sections before the multiplex approach. Therefore, a series of 16 cytological cases of DLBCL was collected. CBs were prepared using the Thermo Scientific™ Shandon™ Cytoblock. Briefly, the samples were fixed before beginning cytoblock preparation. After the concentration of the fixed cells by centrifugation, the cytoblock cassettes had been assembled according to the Thermo Scientific™ Shandon Cytoclip™'s protocol and processed in a standard tissue processor. Sections obtained from CBs were used for FISH analysis.