Zen v 2

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.

The postnatal day (PND) 14 brains were collected and fixed as described above. These brains were transferred into fresh 2% PFA in 0.1 M PB at pH 7.4 and stored at 4°C for up to two months before use. The brains were embedded in 4% agarose and shot with a gene gun (Biorad, 1652451) loaded with homemade bullets consisting tungsten microbeads (Biorad, 1652267) coated with DiI (Invitrogen, D282) at 80 PSI. After gentle washing, the brains were immersed in fresh 2% PFA in 0.1 M PB at pH 7.4 and stored in a dark moisten chamber for 3 days at room temperature to allow dye diffusion. Finally, the brains were sliced at 300uM using a vibrotome (Leica, VT1200) and mounted with coverslip using glycerol. Z-stacks images were acquired immediately using Zeiss LSM-800 confocal microscope and Zen v2.3. The quantification and morphological analysis of spines were done using NeuronStudio v0.9.92. In agreement with literature, the spine shapes were categorized as filopodia, thin, mushroom and stubby (Qiao et al., 2016 (link)). Spines with a neck are classified as thin or mushroom based on the head diameter. Spines longer than 3 mm were classified as filopodia. Spines without a neck are classified as stubby.

Confocal microscopy was used to quantify the expression of periostin in OA cartilage. Images were acquired using ECLIPSE (Nikon) with MetaMorph v7.7 (Molecular Devices) and LSM-880 Confocal Laser Scanning Microscope (Zeiss). The quantification was performed with ZEN v2.3 (Zeiss). Briefly, for human cartilage sections, two randomly chosen fields (60x objective) per slide with two slides per patient were examined. We then selected two fields from each section to evaluate for superficial and deeper zone chondrocytes. About 4–10 chondrocytes per field were used for quantification of signals, which were represented as fluorescent intensity per cell. For mouse knee sections, we selected a site immediately outside of the cartilage damaged area and examined one field (60x) per slide. About 9–13 chondrocytes per field were studied for quantification of signal, represented as fluorescent intensity per cell.