Alexa fluor 488 or 647 secondary antibodies

Target cells were plated at 10,000 cells/well in Packard ViewPlate-96 and incubated 18–24 hours with and without the addition of IFNγ and TNFα (Roche) or resting or BiTE^®-activated T cells. Cells were fixed in 3.7% formaldehyde (ThermoFisher) and stained with anti-ICAM (Abcam) or anti-FAS (ThermoFisher) and Hoechst nuclear dye (ThermoFisher). Goat anti-mouse Alexa Fluor^® 488 or 647 secondary antibodies (ThermoFisher) were used for detection. Untreated cells were used to set fluorescence thresholds for percent positive.

After being anesthetized with euthatal (from Merck) (60 mg/kg), the Nrg1-reporting mice (2 months old) were transcardially perfused with PBS (2 ml/g of body weight), followed by 4% PFA in PBS. Brains were harvested, incubated in 4% PFA overnight, and dehydrated at 4 °C in two steps with 20% and 30% sucrose in PBS. Brains were frozen in OCT (catalog #14-373-65; Fisher) and sectioned into 40 µm slices on a cryostat microtome (Bosch Microm HM550) at − 20 °C. Brain slices were permeabilized with 0.3% TritonX 100 and 5% BSA in PBS and incubated with primary antibodies at 4 °C overnight. The brain slices were not treated with Triton- × 100 when staining with anti-GAD67 antibodies. After washing with PBS for three times, samples were incubated with Alexa Fluor-488 or -647 secondary antibodies (1:1000, ThermoFisher Scientific) for 1 h at room temperature. Samples were mounted with Vectashield mounting medium (Vector) and images were taken by Leica TCS SP8 confocal microscope. The following primary antibodies were used: rabbit anti-NeuN (1:500, Abcam, ab177487), rabbit anti-TH (1:250, Millipore, MAB152), mouse anti-NRGN (1:200, R&D, MAB7947), mouse anti-PV (1:500, Sigma, P3088), mouse anti-GFAP (1:250, Millipore, MAB360); rabbit anti-S100β (1:200, Abcam, Ab52642) and mouse anti-GABA (1:1000, Invitrogen, PA5-32241).