Neurotrace 530 615 red fluorescent nissl stain

In rats 2 and 3, marking lesions were made using 20 µA of anodal current for 10 s; in animals 1 and 4, no marking lesions were made. Animals were transcardially perfused with PBS followed by 4% paraformaldehyde (PFA), brains were cryoprotected in 30% sucrose/4% PFA, and frozen slices of 20 µm (animals 1 and 4) or 40 µm (animals 2 and 3) were cut using a cryostat and stained using either NeuroTrace 530/615 Red Fluorescent Nissl Stain (Thermo Fisher Scientific) or DAPI (Sigma).

Free-floating immunostaining was carried out as follows. Tissue sections were washed twice for 10 min in 0.3% Triton X-100 in PBS (PBS-X) and reacted for overnight to 3 days with primary Abs in PBS-X containing 0.12% λ-carrageenan (035-09693; Sigma-Aldrich) and 1% normal donkey serum (S30-100ML, Merck Millipore) (PBS-XCD). After washing twice for 10 min in PBS-X, the sections were then reacted for 2 − 4 h with secondary Abs in PBS-XCD. The sections were washed twice for 10 min in PBS-X. Some of the sections were counterstained with NeuroTrace 435/455 Blue or NeuroTrace 530/615 Red Fluorescent Nissl Stain (1:100, N21479 or N21482, Thermo Fisher Scientific) in PBS-X. The sections were mounted onto glass slides (Superfrost micro slide glass APS-coated, Matsunami Glass) and coverslipped with 50% glycerol, 2.5% 1,4-diazabicyclo[2.2.2]octane, and 0.02% NaN₃ in PBS (pH 7.4). Endogenous peroxidase activity was quenched with 1% H₂O₂ in PBS for 30 min prior to immunostaining. For multiplex FT-GO IFs, the Ab-POD conjugate was inactivated with 2% NaN₃ in PBS for 4 h after each round of FT deposition. All incubations were performed at 20–25 ºC. Primary Abs (pAbs) and secondary Abs (sAbs) used are listed in Supplementary Table S1 and S2 online.

NeuroTrace 530/615 Red Fluorescent Nissl Stain (1:100, Thermo Fisher, N21482) was used to stain neurons after immunostaining, following the manufacturer’s instructions.

All antibodies and concentrations can be found in Table 1: Antibodies Brain sections were washed with PBS 2 × 5 min then permeabilized with 0.3% PBS-Triton X-100 (PBST) 3 × 10 min. Brains were blocked with a solution of 5% donkey serum and 1% BSA in PBS for ∼2 h at room temperature (RT). Sections were incubated in primary antibody diluted in the above blocking solution for 24–72 h at 4°C with light shaking (50–70 rpm). Sections were then washed in PBS 3 × 10 min before blocking/permeabilizing in a solution of 5% donkey serum in 0.3% PBST for ∼2 h at RT (this step was omitted for MAP2, GAD67, and GFAP immunostainings). Sections were incubated in secondary antibody diluted in 0.3% PBST for ∼2 h at RT with light shaking. After a final wash in PBS 3 × 10 min and a <1-min rinse in deionized water, the sections were placed on SuperFrost Plus slides (Thermo Fisher Scientific) and mounted with VECTASHIELD Antifade Mounting Media with DAPI (Vector Laboratories). For Nissl stains, free-floating sections were rinsed 3 × 5 min in PBS before staining with NeuroTrace 530/615 Red Fluorescent Nissl Stain (Thermo Fisher Scientific) according to manufacturer’s instructions (omitting initial rehydration step). Sections were mounted as noted above.

For immunostaining, brain sections in slides (30 μm) or as floating sections (50 μm) were rinsed once in 1X PBT (PBS + 1% Triton 100X) and incubated in primary antibodies diluted with 1X PBT + 20% normal donkey serum for two nights at 4 °C. After incubation with primary antibodies, sections were rinsed three times with 1X PBT for 10 min and incubated for two hours in the corresponding secondary antibodies (1:800, Jackson-ImmunoResearch). Tissue was then washed three times with 1X PBT for 10–15 min, incubated with DAPI and mounted in Poly aquamount (Polysciences). The following primary antibodies were used: rabbit anti-IBA1 (1:200, Wako), mouse anti-CALB (calbindin,1:200, Sigma-Aldrich), rat anti-CD68 (1:200, Biorad), and rat anti-CD206 (1:200, Bio-Rad). To stain PC somata, the NeuroTrace™ 530/615 Red Fluorescent Nissl Stain was used (NT, 1:80, ThermoFisher Scientific).