Fluoview confocal microscopy

Lung tissues were removed and immunofluorescence staining was performed via a standard protocol. Nonspecific sites were blocked with 3% bovine serum albumin (BSA) in PBS. Anti-S1PR1 (Proteintech Group, Chicago, IL, USA) and anti-CD31 antibodies (Abcam, Cambridge, MA, USA) were used for immunostaining. After incubation with primary antibodies, the tissues were further incubated with fluorescent secondary antibodies (Alexa Fluor dyes). Nuclei were stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma, St. Louis, MO, USA). Finally, confocal immunofluorescence images were captured by Olympus Fluo View confocal microscopy.

Brain sections were washed three times and then permeabilized in TBS with 0.2% Triton-X 100 (TBST; Roche, Switzerland) at room temperature. Sections were blocked in 5% Normal Goat Serum (NGS) in TBST for 1 h at room temperature. Primary antibodies (guinea pig anti-VGlut1 1:2000 [AB5905, Millipore, MA], rabbit anti-PSD95 1:300 [51–6900, Invitrogen, CA], guinea pig anti-VGAT 1:1000 [Synaptic Systems 131 004], rabbit anti-Gephyrin 1:500 [Synaptic Systems 147 002], were diluted in 5% NGS containing TBST. Sections were incubated overnight at 4 °C with primary antibodies. Secondary Alexa-fluorophore (488, 594, and 647) conjugated antibodies (Invitrogen) were added (1:200 in TBST with 5% NGS) for 2 h at room temperature. Slides were mounted in Vectashield with DAPI (Vector Laboratories, CA) and images were acquired on an Olympus Fluoview confocal microscopy using a ×60 oil lens at ×1.64 Zoom.