Autostainer system

Immunostaining was performed for the 10 markers listed in Table 2 in an automated immunostainer (Dako Autostainer System). The antibodies Pou Class 2 homeobox 3 (Pou2f3), absent in melanoma 2 (Aim2), and forkhead box I1 (Foxi1) were tested to validate transcriptomic findings and were evaluated as a percentage of positive cells according to Yamada et al. [24 (link)].Table 2

Antibody sources and dilutions

Antigen	Pretreatment	Dilution	Code Number	Clone	Source
NapsinA (M)	High pH 30 min — 96 °C	1/500	NCL-L-Napsin A	IP64	Leica Biosystems
TTF-1 (M)	High pH 30 min — 96 °C	1/2000	M3575	8G7G3	Dako, Agilent
p40 (M)	High pH 60 min — 96 °C	1/400	API 3079 G3	BC28	Biocare Medical
Chromogranin-A (M)	High pH 60 min — 98 °C	1/100	M0869	Dak-A3	Dako, Agilent
Synaptophisin (M)	High pH 15 min — 96 °C	1/200	M7315	Dak-Synap	Dako, Agilent
Smarca4 (M)	High pH 40 min — 96 °C	1/100	sc-17796	G-7	Santa Cruz
PDL1 (M)	High pH 15 min — 96 °C	1/50	SK006	22c3	Dako, Agilent
Pou2f3 (P)	Low pH 15 min — 96 °C	1/200	HPA019652	Polyclonal	Sigma-Aldrich
Aim2 (P)	Low pH 15 min — 96 °C	1/200	HPA031365	Polyclonal	Sigma-Aldrich
Foxi1 (P)	High pH 30 min — 96 °C	1/500	HPA071469	Polyclonal	Sigma-Aldrich

M, monoclonal; P, Polyclonal; Ki-67, Ki67 index; TTF-1, thyroid transcription factor 1; Pou2f3, POU class 2 homeobox 3; Gli1, glioma-associated oncogene family zinc finger 1; Yap1, Yes1 associated transcriptional regulator; Aim2, absent in melanoma 2; Foxi1, forkhead box I1

For expression analysis by immunohistochemistry, we used the EnVision FLEX High pH (Link) Kit (Agilent-Dako, K800021, Santa Clara, CA, USA) and Dako Autostainer system. Paraffin-embedded tissue sections (3 µm) were deparaffinized, rehydrated, and epitope retrieved by heat induction (HIER) at 95 °C for 20 min and ph 9 (Agilent-Dako) in the Pre-Treatment Module, PT-LINK (Agilent-Dako). Endogenous peroxidase activity was blocked with EnVision™ FLEX Peroxidase-Blocking Reagent (DM821) for 5 min. The sections were incubated with rabbit Anti-PINK-1 polyclonal antibody (BC100-494, NobusBiologicals. Madrid. Spain) at 1:200 dilution for 30 min. The antigen-antibody reaction was detected with the Dako EnVision + Dual Link System-HRP (Agilent-Dako). The signal was detected using diaminobenzidine chromogen as substrate in Dako EnVision™ FLEX /HRP (Agilent-Dako). Counterstaining with hematoxylin was the final step. Negative controls were processed by omitting the primary antibody. Hepatic tissue was used as a positive control [51 (link)]. After the whole process, sections were dehydrated and mounted with a permanent medium (Agilent-Dako mounting medium, CS703). The sections were studied and photographed under a light microscope (Nikon, New York, NY, USA—Eclipse 80i).

For the immunohistochemical analysis of protein levels we used the EnVision FLEX Mini Kit (DAKO, K8024) and Dako Autostainer system. Paraffin embedded tissues (3–5 μm) were deparaffinized, rehydrated, and then epitopes were retrieved by heat induction (HIER) at 95°C for 20 min at pH 6 (DAKO, GV805) in the Pre-Treatment Module, PT-LINK (DAKO).
Sections were incubated with TET2 antibody (Abcam, Ab94580) diluted in EnVision™ FLEX Antibody Diluent (DAKO, K8006) (1:500 dilution) for 30 min after blocking endogenous peroxidase with EnVision™ FLEX Peroxidase-Blocking Reagent (DAKO, DM821). The signal was detected using diaminobenzidine chromogen as substrate after incubation with Dako EnVision™ FLEX /HRP (DAKO, DM822). Sections were counterstained with hematoxylin. Appropriate negative controls were also tested.
After the complete process, sections were dehydrated and mounted with permanent medium (Dako mounting medium, CS703).

FFPE sections were stained with CD31 antibody and then incubated with a commercially available detection kit (EnVision™ FLEX + , Dako, Glostrup, Denmark) in an automated immunostainer (Dako Autostainer System), following the manufacturers’ instructions. Glass slides were then digitalised using the Aperio ScanScope XT (Leica Biosystems, Wetzlar, Germany) slide scanner. Image files were subjected to analyses, using the Aperio ImageScope software, version 11.1.2.760.

FFPE blocks were cut into 3 μm thick sections and stained on a Dako autostainer system with the following antibodies: CD8 (1:100, Dako Cytomation, M7103), CD68 (1:5000, Dako Cytomation, M0814) and CD163 (1:1000, Novocastra, NCL-L-CD163). After standard deparaffinization with xylene and alcohol, the slides were treated with a sodium citrate solution (pH 6.0) at 95 °C for 20 min to retrieve the antigens, and incubated with the mentioned antibodies for 30 min at room temperature. The Dako detection system (FLEX + Mouse, K8002) was used accordingly. Fox P3 (1:25, BioLegend, 320,116) was stained with an automated Ventana BenchMark staining system and the UltraView Universal DAB detection kit (760−500). Counterstaining of all sections was done manually with hematoxylin.

The immunohistochemical analyses of KMT5B protein levels in three GBM samples and paired-normal brain tissue were performed using the EnVision FLEX Mini Kit (DAKO, K8024) and Dako Autostainer system. Briefly, paraffin embedded tissue (3–5 μm) was deparaffinized, rehydrated, and then epitopes were retrieved by heat induction (HIER) at 95°C for 20 min at pH 6.0 (DAKO, GV805) in the Pre-Treatment Module, PT-LINK (DAKO). Sections were incubated with anti-KMT5B antibody (sc-169462, Santa Cruz) at 1:100 dilution in EnVision^TM FLEX Antibody Diluent (DAKO, K8006) for 60 min after blocking endogenous peroxidase with EnVision^TM FLEX Peroxidase-Blocking Reagent (DAKO, DM821). Signal was detected using diaminobenzidine chromogen as substrate after incubation with Dako EnVision^TM FLEX/HRP (DAKO, DM822). Sections were counterstained with hematoxylin. Appropriate negative and positive controls were also tested. After the complete process, sections were dehydrated and mounted with permanent medium (Dako mounting medium, CS703). Images were captured using a Nikon Eclipse Ci microscope equipped with a 20x objective and a camera (Nikon Instruments Europe B. V.).