Axioscan

Following MEMRI, mice were irreversibly anesthetized, exsanguinated, and transcardially perfused with saline followed by 4% phosphate-buffered paraformaldehyde (PFA, pH 7.4), a protocol mildly modified from (Abisambra et al., 2010b (link)). Brain samples were fixed in 4% paraformaldehyde for 24 hr, then cryoprotected by incubating in successive 24 h increments with 10%, 20%, and 30% sucrose gradients as described previously (Jinwal et al., 2010 (link)). Brains were frozen on a temperature-controlled freezing stage, sectioned (25 μm) on a sliding microtome, and stored in a solution of PBS containing 0.02% sodium azide at 4°C.
Free-floating immunohistochemistry was performed as described (Abisambra et al., 2010a (link)). The following antibody dilutions were used: pS262 (Anaspec) 1;10,000; pT231 (Invitrogen) 1:25,000; MC1 (kind gift from P. Davies) 1:2500. IHC sections were imaged using a Zeiss AxioScan (Zeiss, Germany). Image quantification was performed using ImageJ as follows: regions of interest (CA1, CA3, DG, and cortex) in 2–4 coronal sections per mouse were quantified. Then, all images were thresholded identically and intensity was measured for each region of interest. Quantifications shown are mean ± standard error of the mean.

At 7- and 28-dpi, vehicle and DNP-treated mice were anesthetized with a lethal dose of ketamine, exsanguinated using 0.1 M PBS, and perfused using 4% formaldehyde (Thermo Fisher Scientific, Waltham, MA). Spinal cord segments were collected, post-fixed in 4% formaldehyde for 2 h at room temperature, incubated overnight in 0.2 M phosphate buffer, then dehydrated with 30% sucrose (Sigma Aldrich CO., St. Louis, MO) at 4 °C. Spinal cords were placed side by side in cryomolds in sets of 4–5 cords/block in Optimal Cutting Temperature Compound (OCT), and frozen on dry ice. Spinal cords were arranged in a random order while ensuring at least one cord per group was frozen in each block. Sections were cut in the transverse plane at a 10-μm thickness and mounted in serial order, with 100 μm between serial sections on a slide. Prior to immunolabeling all slides were processed using heat-mediated antigen retrieval in 0.1 M sodium citrate buffer (pH 6.0) at 80 °C for 5 min.
Each set of immunolabeling, imaging, and analysis was performed for all animals in a single cohort, using identical staining, imaging acquisition, and analysis settings. Images were acquired using Zeiss Axioscan (model Z1, Carl Zeiss AG., Oberkochen, GE) and analyzed using Halo software (Indica Labs, Albuquerque, NM). Histology and immunohistochemistry was performed by individuals blind to group inclusion as follows.

Synovial tissue samples were frozen in Tissue-Tek OCT medium (Miles, Elkhart, IN) or formalin fixed and paraffin embedded (FFPE).
For immunohistochemistry, antigen retrieval was performed at pH 9 on FFPE sections using Tris-EDTA, 0.05% Tween 20 (10mM Tris Base, 1mM EDTA Solution, 0.05% Tween 20). Sections were stained using anti-FAPα (R&D) and anti-goat Horseradish peroxidase (HRP) (Dako). HRP staining was developed using the ImmPACT DAB Peroxidase HRP Substrate (Vector Labs). Images were acquired using the Zeiss Axio Scan and analysed with Zen lite 2012 software (Zeiss). Number of pixels was quantified and divided by a manually defined tissue area and the average number of pixels per unit area (pixel/UA) was calculated.
For immunofluorescence, acetone fixed frozen sections were incubated with anti-FAPα (F11-24, eBioscience), anti-PDPN (NZ-1.3, eBioscience) and anti-THY1 (Thy-1A1, R&D). These were detected with goat anti-mouse IgG1 FITC, anti-mouse IgG2a TRITC and anti-mouse IgG2b Cy5 (all Southern Biotech). To increase signal from FITC-channel, goat anti-FITC Alexa-488 antibody (Invitrogen) was used. Images were acquired using a Zeiss LSM 510 confocal microscope and ZEN pro 2011 imaging software.