Ab194970

IHC evaluations were performed on 2.5 μm sections of FFPE tissue mounted onto glass slides, dried, and baked at 60 °C for 30 min. Bond RX (Leica Biosystems) Immunostainer was used for automated staining. All slides were dewaxed in Bond dewax solution (product code AR9222, Leica Biosystems). Antigen retrieval was performed in Tris–EDTA buffer based (code AR9640, Leica Biosystems) for 30 min at 95° for anti-CD3 (1:400, Thermo Fisher MA190582); and in citrate buffer based (code AR9961, Leica Biosystems) for 30 min at 100° for anti-CD20 (1:200, Abcam, ab194970), anti-CD31 (1:30, Abcam ab28364), HLA-DR (1:400, Thermo Fisher MA532232;), anti-CD163 (1:400, Thermo Fisher PA578961) (Additional file 1: Table S1). All samples were then incubated with horseradish peroxidase-polymer for 15 min and subsequently visualized using 3,3-diaminobenzidine (DAB) as brown chromogen (Bond polymer refine detection, Leica Biosystems, Ref DS9800) for 10 min. Following these procedures, samples were counterstained with haematoxylin for 5 min, dehydrated, mounted on Pertex (Sakura).

Intracardiac perfusion with DPBS, followed by 4% paraformaldehyde solution, was performed 35 days after tumor implantation. Mouse brains were extracted, frozen, and stored at −80°C. For sectioning, 15 μm cryosections spaced 495 μm apart were cut using a cryostat (Leica CM 1950, Leica, Germany). For immunofluorescence staining, goat anti-human CD20 (3 μg/ml, ab194970, abcam, United Kingdom) was used as a primary antibody, and donkey AF594-conjugated anti-goat antibody (1:200, A11058, Thermo Fisher Scientific, USA) was used as a secondary antibody. DAPI (Sigma-Aldrich, USA) was used for nuclear staining. Slices were observed with a BX60 upright microscope (Olympus, Japan). Tumor area was manually delineated on each section using Axiovision software (Carl Zeiss Microscopy). Total tumor area per slice was multiplied with 0.495 mm, and addition of all values yielded total tumor volume.

For immunodetection of CD20, CD3, and p24, formalin-fixed and paraffin-embedded (FFPE) sections from human lymph nodes of HIV-infected patients were dewaxed and placed in decreasing ethanol concentrations. Heat-induced epitope retrieval was performed by autoclaving at 120 °C for 15 min in a citrate buffer pH 6 (Abcam). Then, the slides were permeabilized in 1X Tris-buffered saline (TBS) (Fisher scientific) with 0.1% Triton X-100 (Sigma-Aldrich) and 1% BSA (Sigma-Aldrich) for 10 min. Subsequently, blocking was added for 2 h with 1X TBS supplemented with 10% normal donkey serum (Jackson Immunoresearch) and 1% BSA. The slides were incubated with primary antibodies overnight at 4 °C: anti-CD20 (goat-polyclonal anti-CD20 antibody, 1/100, Abcam ab194970), anti-CD3 (mouse-monoclonal anti-CD3 antibody, 1/100, Leica Biosystems NCL-L-CD3-565), or anti-p24 (mouse-monoclonal anti-p24 antibody, 1/10, Dako-Agilent M0857), diluted in TBS 1 × −1% BSA. Next, samples were washed and incubated for 1 h with the appropriate secondary antibody: Alexa Fluor 546 donkey anti-goat (Invitrogen, 712-586-150) and Alexa Fluor 647 donkey anti-mouse (Invitrogen, A-31571), counterstained with DAPI (4′,6-diamidino-2-phenylindole-dilactate, ThermoFisher), and mounted with Fluoromount G (eBioscience).