Q path

Deidentified human cremaster biopsies from surgery room (processed within 30 min of the procedure) were embedded in OCT medium (Q-Path, VWR), frozen by immersion in isopentane previously cooled in liquid nitrogen and stored at −80 °C until usage. Cryostat sections (7–10 μm) were stained with haematoxylin and eosin (H&E; Panreac), and mounted with Shandon Consul-Mount mounting media (Fisher Scientific) according to standard procedures. For immunohistochemistry, biopsy sections from OCT blocks were dried; and fixed in 4% Paraformaldehyde aqueous solution (Electron Microscopy Sciences) 10 min at room temperature (RT) or directly blocked with 5% bovine serum albumin (BSA) in PBS for 1 h at RT; and incubated overnight at 4 °C with the primary antibody in 1% BSA solution. Slides were rinsed in PBS containing 0.025% Triton X-100 (Amresco) and incubated for at least 1 h with the appropriate secondary antibody in the same solution used for blocking. The slides were rinsed with PBS containing 0.025% Triton X-100. Nuclei were stained with 10 μg/ml Hoechst solution (Santa Cruz, SC-394039) for 2 min and slides were mounted with Fluoro-Gel mounting media (Electron Microscopy Sciences, Cat #17985–10). Sections were imaged with a Nikon Eclipse 80i fluorescence microscope.

Cultured cells were fixed in 4% formaldehyde (Q Path, VWR Chemicals, Radnor, PA, USA) and prepared for paraffin embedding with the Epredia™ Cytoblock™ Cell Block Preparation System (ThermoFisher Scientific). Sections were stained with hematoxylin and eosin (H&E) using the Leica ST5020-CV5030 Stainer Integrated Workstation (Leica Biosystems, Amsterdam, The Netherlands). The H3F3A p.G34W immunohistochemical stain (RM263, RevMAb, Biosciences, San Francisco, CA, USA) was performed with the Dako Omnis immunostainer (Agilent, Santa Clara, CA, USA) according to the standard diagnostic procedure at LUMC.