Anti parvalbumin α

To evaluate protein levels, protein extracts were prepared as described from dissected forebrain and hippocampal tissue (Murphy et al., 2006 (link)). Primary antibodies: IQSEC2 (N-terminus antibody- 1:100; antiserum UCT 88, a gift from Dr. Randall Walikonis recognizes residues 10-198 of human IQSEC2) and AB_2718685 (C-terminus antibody 1:50 ; Invitrogen recognizes residues 1380 - 1430 of human IQSEC2), guinea pig anti-parvalbumin-α (1:1000; Synaptic Systems, Cat# 195004) , rabbit anti-IQSEC1 (1:500 Biorybt, cat# orb183807), rabbit anti-IQSEC3( 1:500, Thermo Fisher, cat# PA5-25866) anti-glial fibrillary acid protein (GFAP; 1:750; Sigma-Aldrich, Cat# G3893), mouse anti-tubulin (1:2000; Biolegend, Cat# 903401). Signal was detected with corresponding HRP-conjugated secondary antibodies and Immobilon Western Chemiluminescent HRP substrate (Millipore).

Two methods were used for IHC since the IQSEC2 serum antibodies do not work with perfused tissue: Flash-frozen sections (IQSEC2, PV together); PFA perfused sections (PV only). Primary antibodies: rabbit anti-IQSEC2 [1:100; antiserum UCT 88,(Murphy, Jensen, and Walikonis 2006 (link))], chicken anti-IQSEC2 [1:50; antiserum UCT C3(Murphy, Jensen, and Walikonis 2006 (link)),guinea pig anti-parvalbumin-α [1:1000; Synaptic Systems, Cat# 195004], and anti-glial fibrillary acid protein (GFAP) antibody (1:750; Sigma-Aldrich, Cat# G3893). Secondary antibodies: Alexa Fluor 488-conjugated goat anti-mouse, 568-conjugated goat anti-rabbit, and Alexa Fluor 647-conjugated goat anti-chicken IgG (1:500 in blocking buffer; Invitrogen) and Alexa Fluor 555-conjugated goat anti-guinea pig (1:500 in blocking buffer; Abcam)
Parvalbumin immunoreactive neurons were imaged using an automated slide-scanner (Zeiss LSM-800 confocal microscope and Zen v2.3). In a blind study, total counts of parvalbumin immunoreactive cells were made from 3 sections per animal from the left hemisphere using ImageJ (v1.46), from dorsal hippocampus separated into the CA1, CA3 and DG regions. The total counts for the hippocampus included the subiculum. Regions were delineated using clearly visible landmarks and predefined boundaries according to the Allen Brain Atlas.