Ab1822451

Flow synatocytometry was performed as previously described with some modifications [27 (link)]. For extracelluar staining, synaptosomes were stained with primary antibodies (C1q-Abcam, Cambridge, UK, ab1822451) for 30 min at 4 °C and agitated after 15 min. Samples were washed twice with 700 µL of antibody staining buffer (PBS + 5% bovine serum albumin (BSA)) at 13,000× g for 5 min at 4 °C. Secondary antibodies (Goat anti-rabbit 647, Invitrogen, Carlsbad, CA, USA, A21244) were added for 30 min and agitated after 15 min at 4 °C in the dark. Each day, at least one sham and one TBI sample were analyzed; TBI samples were standardized to the sham group. Samples were run in duplicate. Data were collected on an LSRII (BD) and analyzed with Flowjo™ software (v10, FlowJo LLC., Ashland, OR, USA). Overall, 30,000 events were collected for total synaptosomes, and 50,000 events were collected for C1q-expressing terminals.

For immunohistochemistry analysis, tissue preparation, mounting, and blocking were carried out as previously described [27 (link)]. Slides were stained with primary antibodies specific for C1q (Abcam, Cambridge, UK, ab1822451) or PSD-95 (Abcam Cambridge, UK ab13552) overnight, washed three times in Tris-buffered saline (TBS), and stained for the secondary antibody, goat anti-rabbit Alexa-568 (Invitrogen, Carlsbad, CA, USA, A-11011) or goat anti-mouse Alexa-488 (Goat anti-mouse 488 Invitrogen, Carlsbad, CA, USA, A11001). Tissues were fixed using ProLong Gold (Invitrogen, Carlsbad, CA, USA, P36930) and a standard slide cover sealed with nail polish. Two to five images separated by 50–100 µm in the dorsal hippocampus were averaged per animal. For C1q staining, z-stack images were acquired on a Zeiss Imager.Z1 Apotome microscope (Zeiss, Thornwood, NY, USA) controlled by ZEN software (Zeiss 2012, Thornwood, NY, USA) with 200× magnification (C1q). For PSD-95 staining, z-stack images were acquired on a Nikon High-Speed Widefield Confocal microscope (Ti inverted fluorescence; CSU-W1, CSU-W1, Melville, NY, USA) at the UCSF Nikon Imaging Center with 630× magnification.