Triton x 100 pbst

Autoantibody detection was performed via an ELISA using a 96-well microplate coated overnight at 4 °C with SF3B4 recombinant antigens. The remaining binding sites on the microplate were blocked with 20% casein buffer in distilled water. Ten microliters of freshly thawed patient or normal serum samples (diluted 1/250 in 20% casein buffer in PBS) were added to appropriate wells and incubated for 1 h at RT. Each well was washed thrice with PBS with 0.2% Triton X-100 (PBST; Sigma-Aldrich, St.Louis, MO, USA). Diluted horseradish peroxidase (HRP)-conjugated mouse anti-human IgG (Jackson Immuno Research, West Grove, PA, USA) was added to the target wells, and goat anti-mouse IgG was added to the calibrator wells for 1 h at RT. The well was washed with 0.2% PBS-T, and 100 µL of 3,39,5,59-tetramethylbenzidine (TMB; Thermo Fisher, MA, USA) substrate solution was added to each well, followed by incubation for 5 min at RT, and the addition of an equal volume of stopping solution (1 M HCl). The optical density was measured at 450 nm.

Cells were fixed for 15 min using 4% paraformaldehyde (Scharlau, Spain) and washed three times in phosphate buffered saline (PBS) with 0.1% Triton™ X-100 (PBS-T; Sigma Aldrich). Cells were incubated with primary antibodies (Additional file 2: Table S2) diluted in goat immunobuffer (1% goat serum (Gibco), 0.2% triton X-100 and 0.04% thiomersal (Sigma Aldrich) in PBS) at 4 °C overnight, washed three times in PBS-T and incubated with appropriate anti-species fluorescently conjugated secondary antibodies diluted in goat immunobuffer at 4 °C overnight. Cells were washed again in PBS-T and nuclei were counterstained with 20 nM Hoechst 33258 (Sigma Aldrich) for 20 min at room temperature. Images were acquired at 20 x magnification using the ImageXpress® Micro XLS automated fluorescent microscope (Version 5.3.0.1, Molecular Devices, CA, USA). Quantitative analysis of intensity measures and scoring of positively stained cells was performed using the Cell Scoring and Show Region Statistics analysis modules within MetaXpress® software (Molecular Devices). For analysis of microglial morphology by immunocytochemistry (ICC), CD45 staining was thresholded and the Integrated Morphometry Analysis tools Elliptical Form Factor (elongation factor; length/breadth) and Shape Factor (roundness factor; 4πA/P2, P = cell perimeter, A = cell area) were used to determine cell shape.

Mice were deeply anesthetized with isoflurane (2%) and transcardially perfused with phosphate-buffered saline (PBS), followed by 10% neutral buffered formalin (NBF). The animals were decapitated, and the whole heads were incubated overnight in 10% NBF at 4 °C before the fiber optic was removed and the brain was extracted from the skull. Brains were incubated in 10% NBF for up to 4 h followed by 30% sucrose (w/v in PBS) for cryoprotection. 50 µm coronal brain sections were obtained using a cryostat (Leica Biosystems, ON, CA, Cat. #CM1850 UV). The sliced brain sections were collected in a staggered fashion and placed into 3 consecutive wells. Sections were rinsed before and between incubations with 0.1 M PBS. Sections were incubated in blocking solution (5% donkey serum in PBS with 0.5% Triton-X 100 (PBST), Sigma-Aldrich, St. Louis, MO, USA) for 1 h and blocking solution (5% donkey serum in 0.3% PBST) was used in subsequent antibody incubations. Controls were included where the primary antibody was omitted to check for non-specific binding of the secondary antibodies. Free-floating brain sections were then mounted onto Superfrost^TM slides in VECTASHIELD® PLUS Antifade Mounting Medium (Vector Laboratories Inc., Newark, CA, USA, Cat. #VECTH190010) and cover-slipped. All primary and secondary antibodies, along with their conjugates, are presented in Supplementary Table 1.

To visualize phagocytosis under conditions of proinflammatory LPS and PAL stimulation and its modulation by 5-AZA and SAM methylation modulators, we used confocal microscopy. SIM-A9 cells were seeded on sterile coverslips in six-well plates covered with 0.01% poly-L-lysine solution (Sigma, p4707). After treatment, cells were fixed by adding 4% paraformaldehyde (PAF) (Sigma, 158127) for 10 min, washed three times with 1 × PBS, and were permeabilized with PBS solution 1 × + 0.1% Triton X-100 (PBST) (Sigma, × 100) for 10 min. Next, the cells were blocked in PBST + 10% goat serum (GIBCO, 16210064) for 30 min and washed three times with 1 × PBS. Microglia immunodetection was performed by overnight primary antibody anti-mouse Iba-1 (1:200) (Abcam, ab178847) incubation at 4°C followed by goat anti-rabbit IgG coupled to Alexa Fluor 546 (1:1000) (Invitrogen, A-11035). Finally, cells were washed 3 × with 1 × PBS and mounted in VECTASHIELD mounting medium with DAPI (Vector Laboratories, H-1000-10). Fluorescent signals were detected by confocal-laser microscopy using an Olympus BX61W1 microscope with an FV1000 module with diode laser. Finally, the images were processed with ImageJ software.

GBM, GIC0222, and TGS01 cell lines were plated on a 24-well plate containing BD BioCoat poly-L-lysine cellware 12-mm round coverslips with 10% FBS/DMEM and incubated for 24 h.
The coverslips were rinsed twice with phosphate-buffered saline (Thermo Fisher Scientific) and placed in 4% paraformaldehyde phosphate buffer solution (PFA; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) for 15 min.
Blocking was performed in PBS containing 0.1% Triton X-100 (PBST; Sigma-Aldrich) with 1.5% goat serum for 1 h at room temperature with shaking. The coverslips were incubated with the primary antibody Lp2, diluted to 1 mg/mL in blocking solution for 1 h at room temperature with shaking, followed by three washes with PBS. The coverslips were stained using a secondary antibody, Alexa Fluor 488 Goat Anti-Rat IgG (HþL; Thermo Fisher Scientific), and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) solution (Dojindo, Kumamoto, Japan) at 1:200 dilution in the blocking solution for 30 min in the dark, followed by three washes with PBST. They were then mounted onto slides. Stained cells were observed under a fluorescence microscope (Keyence BZ-X710, Osaka, Japan), and pictures were taken.