Srp0292

Free floating mouse brain sections were incubated with 300 ng of active human cathepsin-S (SRP0292, Sigma-Aldrich), in activation buffer containing 1.8 mm DTT, 1.8 mm EDTA, 1% BSA, 12 mm citric acid, and 43 mm Na₂HPO₄ at 37°C for either 3 h or 24 h. Control sections were incubated in activation buffer (1.8 mm DTT, 1.8 mm EDTA, 1% BSA, 12 mm citric acid, and 43 mm Na₂HPO₄) at 37°C in parallel. Following cathepsin-S incubation, sections were labeled with WFA and WFA+ PNNs were quantified in the hippocampus as described above.

Free-floating tissue sections were carried through antigen retrieval in citric acid buffer (0.1 m citric acid and 0.2 m Na₂HPO₄), heated to 80°C for 30 min, and incubated in biotinylated WFA lectin (catalog #B-1355, Vector Labs) or the mouse monoclonal primary antibody anti-cathepsin-S (1:500, sc-271619, Santa Cruz Biotechnology Inc.) for 48 h, and subsequently in biotinylated secondary antibody (horse anti-goat IgG; 1:500; Vector Labs), followed by streptavidin conjugated with horse-radish peroxidase for 2 h (1:5000 μl, Zymed), and, finally, in nickel-enhanced diaminobenzidine/peroxidase reaction (0.02% diaminobenzidine, Sigma-Aldrich, 0.08% nickel-sulfate, 0.006% hydrogen peroxide in PB). All solutions were made in PBS with 0.2% Triton X-100 (PBS-Tx) unless otherwise specified. Immunostained sections were mounted on gelatin-coated glass slides, coverslipped, and coded for blinded quantitative analysis. All sections included in the study were processed simultaneously within the same session to avoid procedural differences. Omission of the primary or secondary antibodies did not result in detectable signal, and preabsorption of mouse anti cathepsin-S with 300 nanograms of active human cathepsin-S (SRP0292, Sigma-Aldrich) did not result in detectable immunolabeling signal.