M7240

Tumor sections were formalin-fixed and paraffin embedded. H&E or IHC staining was performed using a Discovery XT automated immunostainer (Ventana Medical System). After dewaxing, deparaffinization, and rehydration, Tris-EDTA buffer was used for antigen retrieval. The sections were immunostained for PCNA (GTX #100539, 1:500, GeneTex, USA) and Ki67 (Dako #M7240, 1:150, DAKO/Agilent, Santa Clara, CA), and subsequently counterstained with hematoxylin.

ATRT tissues from patient, PDX model and orthotopic xenograft mice model were embedded in paraffin after fixing in 4% paraformaldehyde. Normal brain tissue slides (NBP2-50617, Novus Biologicals, Centennial, CO, USA) were used as controls. Tumor sections (5 µm in thickness) were deparaffinized twice in xylene for 10 min each and twice in ethanol for 2 min each. Sections were fixed in 4% paraformaldehyde for 20 min, air dried and blocked with 10% goat serum containing 1% BSA in PBS at room temperature for 30 min. The following primary antibodies were used for IHC: anti-INI1 (1:300, 612110, BD Biosciences, San Jose, CA, USA), anti-PSMD4 (1:100, HPA038807), anti-PSMB4 (1:25, HPA006700, Atlas Antibodies, Stockholm, Sweden), anti-p53 (1:600, MS-187-P0, Thermo Scientific, Fremont, CA, USA) and anti-Ki67 (1:800, M7240, Agilent, Santa Clara, CA, USA). Appropriate positive and negative controls were included in the IHC staining. Images were captured and analyzed by TissueFAXS (TissueGnostics, Vienna, Austria) at the Taipei Medical University (TMU) Core Facility Center (Taipei, Taiwan).

Tumour tissue was harvested and immediately fixed in 10% neutral buffered formalin at 4 °C overnight before dehydration and paraffin embedding. Antibodies used for IHC were anti-ER (M7047, 1:300, Agilent) and anti-Ki67 (M7240, 1:400, Agilent). Primary antibodies were detected using biotinylated IgG secondary antibodies (Agilent, 1:400), using streptavidin-HRP (Agilent) for amplification of signal followed by the addition of 3,3′-diaminobenzidine (Sigma) substrate. Images were scanned using Leica Aperio Slide Scanner (Leica Biosystems) and analysed using QuPath software to differentiate tumour tissue from stroma and necrosis, and to quantify Ki-67 positivity in tumour tissue.

Lymphoma tumors from mice were processed by fixation in formalin followed by paraffin embedding. Prior to staining, tumors were deparaffinized in alcohol gradient, washed, and then subjected to antigen retrieval for 45 min in a steamer using Target Retrieval Solution (1×; pH 9.0, Dako). Sections were left for 20 min to cool down to room temperature, washed, and incubated in 3% H₂O₂ for 15 min to block endogenous peroxidase activity. Sections were then blocked for 30 min at room temperature in serum-free blocking solution (Dako). The primary antibody, diluted in blocking buffer, was added for overnight incubations at 4 °C. Primary antibody dilutions were 1:50 for ALK (M719501-2, Dako) and pSTAT3^Y705 (4113, Cell Signaling); and 1:75 for pIGF-IR^Y1161 (ab39398, Abcam, Cambridge, MA) and Ki-67 (M7240, Dako). Sections were washed three times and incubated for 30 min with the secondary antibody Dako Envision+Link System-HRP. Signals were developed using 3,3’-diaminobenzidine tetrachloride substrate, and hematoxylin was used for counterstaining.

FFPE samples were embedded in paraffin after fixation with 10% formaldehyde. 4 μm slices were cut from FFPE samples, mount on coated slides, and dried for 3 h at 42 °C. After deparaffinization, rehydration, and wash, antigen activation was performed by heating in boiled citrate buffer solution (pH 6.0). Samples were incubated for 1 h at 20 °C in a 4% Donkey Serum/1% BSA/Tris-buffered saline with Tween 20 (TBS-T) solution followed by 4 °C overnight incubation with primary antibodies in 2% Donkey Serum/TBS-T. After five times washes with TBS-T, samples were exposed to secondary antibodies for 1 h at 20 °C. For GJA4 staining, samples were incubated for 1 h in biotinylated antibody, followed by incubation in streptavidin secondary antibody in 2% Donkey serum/TBS-T for 1 h at 20 °C. Three more washes with TBS-T were followed by incubation with the TOTO®-3 nuclear stain. Images were recorded on the ZEISS Axio Imager M1 (Carl Zeiss, Oberkochen, Germany). Following antibodies were used: GJA4 (Abcam ab181701, 1:500), CD31 (R&D Systems AF3628, 1:200), αSMA (Abcam ab21027, 1:200), Ki-67 (Dako, M7240, 1:200), Donkey anti-Rabbit IgG, biotin-SP (Merck Millipore AP182B, 1:500), Streptavidin, Alexa Fluor 488 (Thermo Fisher Scientific S11223, 1:200), Donkey anti-Goat IgG, Alexa Fluor 546 (Invitrogen A-11056, 1:200), TOTO®-3 (Thermo Fisher Scientific T3604, 1:500).