Brightvision plus kit

Formalin-fixed and paraffin-embedded tumor tissue was deparaffinized in Roti-Histol (Carl Roth) and rehydrated in isopropanol before sections were incubated in a pressure cooker at 121 °C for 5 min in a coplin jar containing Dako target retrieval buffer pH 6 (Dako, Hamburg, Germany). Sections were cooled down to room temperature, blocked for 15 min in PBS supplemented with 5% BSA and incubated overnight at 4 °C with a rabbit anti-H3.3 G34W antibody (RevMab Biosciences, San Francisco, CA, USA) diluted 1:200 in PBS supplemented with 1% BSA. Signals were detected using a BrightVision plus kit (VWR International, Bruchsal, Germany) according to the manufacturer’s instructions. For visualization, the red substrate ImmPACT Vector Red (Linaris, Dossenheim, Germany) was used. After 30 min of staining, the samples were counterstained with methyl green, mounted with NeoMount (Merck-Millipore, Darmstadt, Germany), and photographed using a Keyence BZ-X800 microscope (Keyence, Neu-Isenburg, Germany).

Formalin-fixed, paraffin-embedded tissue sections were deparaffinised in Roti–Histol (Carl Roth) and rehydrated with isopropanol. Antigen retrieval was performed using Dako target retrieval buffer pH 6 (Dako, Hamburg, Germany) in a pressure cooker at 121 °C for 5 min. Sections were blocked with PBS supplemented with 5% BSA and incubated with the primary antibody specific for cleaved caspase-3 (Cell Signaling, Leiden, The Netherlands) diluted 1:250 in PBS/1% BSA overnight at 4 °C. Detection was carried out using the BrightVision plus kit (VWR International, Bruchsal, Germany) according to the manufacturer’s instructions. ImmPACT Vector Red (Linaris) was used as substrate. Samples were counterstained with Gill´s haematoxilin (Santa Cruz) and mounted with NeoMount (VWR International).