Dako target retrieval buffer ph 6

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).

Red staining of TRAP of GCTB tissue sections was performed as described in our recent publication.^{46 (link)} For SOX2 and Oct-4 staining, formalin-fixed, paraffin-embedded GCTB tissue sections were de-paraffinized in Roti-Histol (Carl Roth GmbH, Karlsruhe, Germany) and rehydrated in isopropanol. Antigen retrieval was performed using Dako target retrieval buffer pH 6 (Dako, Hamburg, Germany). The mouse mAbs against SOX2 (Merck Millipore) and OCT4 (Santa Cruz, Heidelberg, Germany) were used as primary antibodies. NBT/BCIP was used as a chromogen. For c-Met staining of GCTB patient tissue or GCTB-derived stromal cell xenografts, endogenous biotin was blocked using the Avidin/Biotin Blocking Kit (Vector, Burlingame, CA, USA) according to the manufacturer's instructions. Endogenous peroxidase was blocked by 0.3% H₂O₂ in methanol. A rabbit pAb against human c-Met (Abcam) was used as the primary antibody. Biotinylated goat anti-rabbit IgG (Vector) was used as the secondary Ab. The signal was amplified using the ABC Elite Kit (Vector). AEC was used as a chromogen. Samples were counterstained with hematoxylin (Mayer, Sigma-Aldrich, Steinheim, Germany) and mounted using Pro Tags Aqua mount (Quartett, Berlin, Germany). Omission of the primary Ab served as a negative control.

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).