Immunohistochemical Analysis of C1q and PSD-95

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.