Dapi dye

CLL cells were fixed with 2% paraformaldehyde at 37°C, permeabilized and kept overnight at −20°C. After being washed with PBS supplemented with 2% FBS (Invitrogen), the cells were incubated for 1 h in the presence of rabbit anti-PTX3 and mouse anti-STAT3 antibodies. After washing with PBS, the cells were incubated with Alexa Fluor 488-labeled anti-rabbit and Alexa Fluor 647-labeled anti-mouse antibodies (Supplemental Table SII). After being washed with PBS, the cells were suspended in DAPI dye (Invitrogen) for 15 min and then washed in PBS to remove the unbound dye. Finally, the cells were transferred onto µ-slide VI chamber slides (Ibidi, Fitchburg, WI), imaged using an upgraded FV1000 confocal microscope, and visualized using the FluoView software (Olympus, Tokyo, Japan).

Mice were deeply anesthetized with ketamine/xylazine and transcardially perfused with cold phosphate-buffered saline (PBS) followed by cold 4% paraformaldehyde (PFA) in 1 × PBS. The brains were dissected and post-fixed in 4% PFA in PBS at 4°C overnight. Free-floating vibratome coronal sections (35 μm) were cut and incubated in a blocking solution containing 10% normal donkey serum, 0.2% Triton-X 100, 3% bovine serum albumin, and 0.02% sodium azide in 1 × PBS for 2 hrs at room temperature (RT). Sections were labeled with primary anti-c-Fos antibody (1:400; sc-52; Santa Cruz Biotechnology, USA) in the blocking solution at 4°C overnight, followed by the Alexa488-conjugated (1:1000; Invitrogen, ThermoFisher Scientific, USA) secondary antibody for 1 hr at RT. Slices were incubated for 20min in 1 × DAPI dye (Invitrogen, ThermoFisher Scientific, USA) in PBS at RT to label cell nuclei. Samples were then washed 4 × 15 min in PBS with 0.1% Triton-X 100. Immunolabeled brain sections were mounted onto glass slides using ProLong Gold anti-fade reagent (Invitrogen, ThermoFisher Scientific, USA) and stored at −20°C. Images of the cortical and hippocampal areas from the stimulated and unstimulated sides of the brain were acquired using a high-resolution multi-channel (sequential) scanning confocal microscope (LSM 510, Zeiss, Germany), using a 10× air objective (NA 0.45).