Guinea pig anti synaptophysin

Following BV2-neuronal coculture, cells were fixed in 4% paraformaldehyde, permeabilized for 20 minutes in 0.25% Tween-20, and immunostained with a microglial marker rabbit anti-Iba1 (1:1000, Wako, 019-19741), and guinea pig anti-synaptophysin (1:1000; Synaptic Systems) for 2 hours at room temperature, followed by secondary antibodies (donkey anti-rabbit 488 (1:500; Invitrogen, A21206); goat anti-guinea pig 555 (1:500; Invitrogen, A21435) for one hour at room temperature. After washing the slides with Tris-buffered saline with Tween-20 (TBST), slides were stained with DAPI (4′,6-diamidino-2-phenylindole, a blue-fluorescent DNA stain). Cells were imaged at 20x using Zeiss Axioplan II microscope with six representative images captured and quantified per coverslip in a blinded fashion. The local density of neuronal processes surrounding BV2 cells was quantified in ImageJ by measuring the synaptophysin staining density within the BV2 cell and surrounding area (defined by BV2 nuclei (DAPI) area and adjacent area extending out from the DAPI image perimeter using the ImageJ function “Mask of Image Points” (version 1.1) with settings “add mask points within distance” = 1 micron. We used the expanded DAPI area to localize BV2 cells rather than the Iba1 signal because Iba1 staining intensity varied from cell to cell.