Clone 6f2

Unstained 3-μm-thick slides of formalin-fixed, paraffin-embedded tissues were obtained and submitted for immunostaining with an automated stainer (Dako Omnis, Glostrup, Denmark). The following primary antibodies were used: CD56 (pre-diluted, clone 123C3, Dako, Glostrup, Denmark), Glial Fibrillary Acidic Protein (GFAP) (1:200, clone 6F2, Dako, Glostrup, Denmark), Olig2 (1:500, clone OLIG2, Sigma-Aldrich, Saint-Louis, USA), vimentin (1:800, clone V9, Dako, Glostrup, Denmark), neurofilament (1:100, clone NF70, Dako, Glostrup, Denmark), NeuN (1:1000, clone A60, Sigma-Aldrich, Saint-Louis, USA), synaptophysin (1:150, clone Synap, Dako, Glostrup, Denmark), EMA (1:200, clone GM008, Dako, Glostrup, Denmark), CK18 (1:200, clone 6F2, Dako, Glostrup, Denmark), smooth muscle actin (1:4000, clone 1A4, Dako, Glostrup, Denmark), NFκB (1:6000, clone D14E12, Cell Signaling Technology, Danvers, USA), L1CAM (1:500, clone UJ127.11, Sigma-Aldrich, Saint-Louis, USA), and Ki-67 (1:200, clone MIB-1, Dako, Glostrup, Denmark). Reticulin staining was performed using the Reticulin silver plating kit according to Gordon & Sweets (Merck Millipore, Guyancourt, France). External positive and negative controls were used for all antibodies and staining.

Formalin-fixed paraffin-embedded tissue (FFPE) samples from the superior frontal gyrus (Brodman area 9 or 8) were processed and stained with hematoxylin and eosin following standard laboratory procedures. Immunohistochemistry with antibodies to human glial fibrillary acidic protein (GFAP; 1:200, clone 6F2; DakoCytomation, Glostrup, Denmark), human leukocyte antigen DR, (HLA-DR, DP, DQ; 1:200, mouse clone CR3/43; DakoCytomation), CD8 (1:100, clone SP239; Spring Bioscience, Pleasanton, USA), and SARS-CoV-2 nucleocapsid (1:1000; clone 4A8, Synaptic Systems, Goettingen, Germany) was performed on a Ventana benchmark XT autostainer following the manufacturer's recommendations. The quality of the immunohistochemical stains was assessed by on-slide positive controls for all antibodies. For semiquantitative assessment of cytotoxic T lymphocyte infiltration, cells with positive CD8 staining were counted per high-power field (HPF) of 0.5 mm². Infiltration was categorized as none, mild (1 to 9 cells per HPF), moderate (10 to 49 cells per HPF), or severe (≥50 cells per HPF).

Double immunofluorescence stainings were carried out manually on selected tumour specimens using antibodies against Sox9 and β-catenin, Olig2 or GFAP (monoclonal mouse anti GFAP; 1:500; clone 6F2; Dako; Glostrup, Denmark) as well as Sox2 and Olig2 or GFAP. After dewaxing the slides, antigen retrieval by microwave pre-treatment was performed in citrate-buffer at pH 6. To prevent unspecific antibody binding, the slides then were incubated for two hours with a blocking solution containing PBS with foetal calf serum (5%; Biochrom AG; Berlin, Germany), goat serum (3%; Millipore; Temecula, CA, USA) and Triton ×100 (0,1%; Sigma-Aldrich; Steinheim, Germany). Primary antibodies were diluted in the blocking solution and incubated overnight at 4 °C. Carbocyanine 2 (Cy2) goat anti-mouse or goat anti-rabbit (1:100; green; Dianova; Hamburg, Germany) and Cy-3 goat anti-rabbit or goat anti-mouse secondary antibodies (1:100; red; Dianova; Hamburg, Germany) served as fluorescent markers, while cell nuclei were counterstained with Hoechst 33342 (Sigma Aldrich; Steinheim, Germany) at a concentration of 500 ng/ml for 5 min at room temperature. Slides were analysed using an Olympus BX-51 fluorescent microscope (Olympus; Hamburg, Germany) equipped with an F-View II CCD camera (Soft imaging systems; Stuttgart, Germany).