Biotinylated anti mouse antibody

The following primary antibodies were used in the present study: mouse anti-Ttyh1 (1:200, #WH0057348M4, Sigma), rabbit anti-Synaptotagmin 1 (1:100, #105102, Synaptic Systems), rabbit anti-Synaptoporin (1:500, #102002, Synaptic Systems), rabbit anti-Neuronal Class III β-Tubulin (1:750, #PRB-435P, Covance), rabbit anti-LAMP1 (1:500, #ab24170, Abcam), rabbit anti-GRP78 (1:200, #ab21685, Abcam), rabbit anti-Calnexin (1:40, #ab22595, Abcam), rabbit anti-GM130 (1:200, #ab52649, Abcam), rabbit anti-TGN46 (1:500, #ab16059), rabbit anti-Clathrin (1:500, #ab21679, Abcam), rabbit anti-Rab5 (1:500, #ab18211, Abcam), rabbit anti-Rab11 (1:100, #71-5300, Invitrogen), rabbit anti-APPL2 (home made, [20 (link)]), rabbit anti-Olig2 (1:500, #AB9610, Chemicon Millipore), rabbit anti-Iba1 (1:1,000, #019-19741, Wako), mouse anti-GFAP-Cy3 (1:1,000, #C9205, Sigma). Anti-IgG mouse (1:200, #553447, BD Pharmingen) anti-IgG rabbit (1:200, #ab46540, Abcam) were used as controls to test specificity of immunostainings. The following secondary antibodies were used in the present study: goat anti-mouse conjugated with Alexa Fluor 488 (1:200, #A11001, Molecular Probes), goat anti-rabbit conjugated with Texas Red (1:200, #T2767, Molecular Probes), horse anti-mouse conjugated with Texas Red (1:200, #TI2000, Vector Laboratories), and anti-mouse biotinylated antibody (1:200, #BA2001, Vector Laboratories).

Tumour biopsy sections (4–5 μm) were de-paraffinised and rehydrated, with antigen retrieval in citrate buffer (pH 6) and unspecific binding sites blocked with 1.5 % horse serum in PBS. The sections were then stained with mAb p16^INKA4a (clone: JC8, dilution 1:100, Santa Cruz Biotech, Santa Cruz, CA, USA) at +8 °C overnight, before incubation for 45 min. with biotinylated anti-mouse antibody (dilution 1:200, Vector Laboratories, Burlingame, CA, USA). Alternatively, the slides were stained with the CINtec® p16 Histology (805–4713), Ventana Medical Systems Inc., Arizona, USA by following the same protocol with the exception of incubation with the antibody for 1 h at room temperature. For antigen detection, the avidin–biotin–peroxidase complex (ABC) kit (Vectastain, Vector Laboratories, Burlingame, CA, USA) was used. Slides developed in chromogen 3’-diaminobenzydine (DAB) (Vector Laboratories, Burlingame, CA, USA) and counterstained with haematoxylin were then washed and dehydrated, and the cover mounted using VectaMount permanent mounting media (Vector Laboratories, Burlingame, CA, USA). P16 staining was regarded as positive if >70 % of the tumour cells were strongly p16-positive [28 (link), 29 ].

30µm thick  sagittal free-floating sections were incubated with anti-Iba1 (1:500, cat#019-19741, Wako, Richmond, VA, USA), 6E10 (1:1000, cat#9320-500, Covance, Princeton, NJ, USA), or C1q (1:1000, cat#ab182451, Abcam, Cambridge, MA, USA) antibodies. Sections probed with Iba1 and 6E10 antibodies were incubated with anti-rabbit Alexa 488 (1:400, cat#A-11008, Thermo Fisher Scientific, Grand Island, NY, USA) and anti-mouse Alexa 594 (1:400, cat#A-11005, Thermo Fisher Scientific) secondary antibodies. Sections probed with C1q were incubated with biotinylated anti-mouse antibody (1:1000, BA-9200, Vector laboratories, Burlingame, CA, USA) and developed with Vectastain ABC Kit (PK-4000, Vector Laboratories) (see Suppl. methods for details). Images were acquired using an Olympus BX61 microscope or a Pannoramic 250 digital scanner (3DHISTECH, Budapest, Hungary).
For measuring percentage of 6E10-immunoreactive areas, 6E10-immunolabeled sections were thresholded and the percentage of 6E10 positive area was calculated using Fiji (v2.0.0) and the “measure” function. For measuring microglial number, Iba1 positive cells in the region of interest (ROI) were manually counted and number was normalized to the area of the ROI. ROI (PFC and hippocampus) were determined and outlined by manual tracing.

Cultured cells were treated with 40 μM 5-bromo-2′-deoxyuridine (BrdU) for 18 h before fixation. The cells were fixed with absolute cold ethanol for 15 min. Subsequently, they were incubated with 2 N HCl for 30 min at RT and then incubated with 0.6% hydrogen peroxide in absolute methanol for 30 min at RT. After blocking with BlockAce for 30 min at RT, the cells were incubated with a mouse anti-BrdU antibody for 60 min. The dishes were rinsed with PBS and subsequently incubated with a biotinylated anti-mouse antibody (Vector Laboratories, Burlingame, USA) for 30 min at RT. Next, the cells were incubated with an avidin-biotin complex solution (VECTASTAIN ABC kit; Vector Laboratories) and treated with 3,3-diaminobenzidine for color development. The number of cells with BrdU-positive nuclei was counted to determine the LI.

Patient material for the tissue microarray was derived from paraffin embedded GBM biopsies obtained from the Department of Pathology, Haukeland University Hospital, Bergen, Norway. The project (number 2017/2505) was approved by the Regional Ethics Committee (Bergen, Norway). Control tissues (brain, liver and tonsil) were derived from autopsy material. The microarray included 61 primary GBM and 49 recurrent GBM biopsies, plus 12 biopsies from normal tissues (four each from brain, tonsil and liver). Immunohistochemical staining was carried out as described previously [30 ] using galectin-1 antibody (sc-166618; Santa Cruz, Santa Cruz, CA, USA) at a dilution of 1:200 followed by a biotinylated anti-mouse antibody (Vector Laboratories, Burlingame, CA, USA) at a dilution of 1:1100. Galectin-1-positive cells were counted via automated counting, as described previously [31 (link)].