Krystalon

Sections were brought to room temperature in 0.1 M phosphate buffer (PB; pH 7.4), then exposed to citrate buffer (pH 5.6–6.0) at 90–95°C for 10 min, followed by a 5-min PB wash (37°C), 3 min in a solution of 0.28% pepsin in 400 ml 0.1 M HCl at 37°C, and three 5-min washes in PB at room temperature. Non-specific binding was blocked with 3% normal donkey serum (Jackson Labs) and 0.5% Triton X-100 in PB (“block”) for 1 h at room temperature, followed by 24–48 h exposure to sheep anti-BrdU (1:239, Capralogics) in block at 4°C. Sections were again rinsed in PB and then incubated overnight in biotinylated donkey anti-sheep IgG in PB (1:200, Vector Labs), followed by overnight incubation in streptavidin-conjugated Alexa 488 in PB (1:800, ThermoFisher Scientific) for visualization of BrdU. The next day, sections were washed with PB, blocked for one hour, and incubated overnight in mouse anti-Hu primary antibody at 4°C (1:200 in 3% block; Invitrogen). After three 5-min PB rinses at room temperature, tissue was exposed to donkey anti-mouse IgG conjugated to Cy-3 in PB (1:80; EMD Millipore) for 1 h for visualization of Hu. Finally, sections were washed, dehydrated with ethanols, delipidized with xylenes, and cover slipped with Krystalon (Millipore-Sigma).

Wild type and Ildr1^-/- mice were transcardially perfused with ice-cold heparinized PBS, followed by freshly depolymerized 3.5% paraformaldehyde in PBS. Tissues were dissected and post-fixed in 3.5% paraformaldehyde, cryopreserved sequentially in 10%, 20%, and 30% sucrose in 0.1M phosphate buffer, pH 7.4, embedded in Tissue-Tek® OCT compound (VWR, Radnor, PA), and cryosectioned at 8–10 μm. Sections were post-fixed in 0.2% glutaraldehyde, 5 mM EGTA in PBS for 10 min at 4°C, stained with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) using the protocol developed by Kishigami and colleagues [25 (link)]. After X-gal color development, sections were rinsed and counterstained with Nuclear Fast Red stain (Millipore Sigma, St. Louis, MO) for 10 min, followed by two washes in PBS. Slides were dehydrated by passing them sequentially through 70%, 95%, and 100% ethanol (2 times, 30 sec each), they were cleared in xylene (twice, 5 min each) and mounted in Krystalon (Millipore Sigma).

Sections were brought to room temperature in tris buffered saline (TBS), followed by a 10-min wash in fresh TBS. Sections were then incubated for 30-min in a H₂O₂ solution (97% TBS, 1% methanol, 2% of 3% retail H₂O₂) to eliminate endogenous peroxidases. After three 5-min TBS rinses, non-specific binding was blocked with 5% normal horse serum (Jackson ImmunoResearch Laboratories Inc.) and 0.5% Triton X-100 (Sigma-Aldrich) in TBS for 30-min at room temperature. This was followed by exposure to anti-DCX antibody (goat polyclonal IgG, Santa Cruz Biotechnology; sc-8066, 1:150; or rabbit polyclonal IgG, Abcam; ab18723, 1:1000) in the same blocking buffer overnight at 4˚C. After three 5-min TBS rinses, sections were incubated for 3 h in biotinylated horse anti-goat (1:200, Vector Labs) or biotinylated goat anti-rabbit secondary antibody (1:200, Vector Labs) in TBS, rinsed again, and exposed for 1 h to an avidin-biotin complex (Vector Labs). Sections were rinsed in TBS and then reacted in a solution of 0.04% of 3,3’-diaminobenzidine tetrahydrochloride (DAB, Vector Labs) with nickel until the tissue changed color (~3–10 min). Following three 5-min TBS rinses, sections were dehydrated in ethanols, delipidized in xylenes, and cover slipped with Krystalon (Millipore-Sigma).