Alexa fluor 488 or 594 conjugated phalloidin

Cells were fixed with 4% paraformaldehyde (PFA) for 8 min at room temperature (RT). After fixation, 0.1% Triton X-100 (Sigma-Aldrich) was applied for permeabilization. Cells were incubated with primary antibodies for 1 h at RT. Cells were incubated with Alexa Fluor 488- or 594-conjugated anti-mouse or anti-rabbit secondary antibodies (Invitrogen; 1:1000 dilution) for 1 h at RT. For staining actin filaments, Alexa Fluor 488- or 594-conjugated phalloidin (Invitrogen) was used according to the manufacturer’s protocol. For immunofluorescence staining of the mouse tumor, tumor tissues were fixed in 4% PFA overnight in 4 °C, dehydrated in 30% sucrose solution, and embedded in tissue freezing medium (Leica). Cryosections were blocked with 3% donkey serum in PBST (0.3% Triton X-100 in PBS) and then incubated at 4 °C overnight with primary antibodies. The samples were washed five times with PBS, followed by incubation with secondary antibodies (Invitrogen; 1:1000 dilution) for 2 h at 4 °C. Fluorescence images were acquired using a DeltaVision Spectris Imaging System (Applied Precision) and LSM800 confocal microscope (Carl Zeiss).

mBMDCs were seeded on gelatin-coated glass coverslips for 0.5–1 h, fixed with 4% PFA in PBS for 15–20 min at RT and permeabilized using 0.1% Triton X-100 in PBS. To visualize host cell F-actin, cells were stained with Alexa Fluor 488- or 594-conjugated phalloidin (Invitrogen). To probe GABA-A R subunits, cells were incubated with rabbit anti-GABA-A R α3 polyclonal antibody, rabbit anti-GABA-A R α5 polyclonal antibody, rabbit anti-GABA-A R ρ1 polyclonal antibody (all from Alomone labs, Jerusalem, Israel), mouse anti-GABA-A R β3 monoclonal antibody (NeuroMab, UC Davis, CA, US) and for NKCC, with mouse anti-NKCC1/2 monoclonal antibody (clone T4, Developmental Studies Hybridoma Bank, DSHB, Iowa, USA) ON at 4°C. Following staining with respective Alexa Fluor 488- or 594-conjugated secondary antibodies (Thermofisher) and DAPI, coverslips were mounted and imaged by confocal microscopy (LSM 780 and LSM 800, Zeiss).