Thionin

The procedure for Nissl-stained sections followed standard laboratory protocols (Lavenex et al., 2009 (link)). Sections were removed from 10% formaldehyde solution, thoroughly washed for 2 × 2 h in 0.1 M PB (pH 7.4), mounted on gelatin-coated slides from filtered 0.05 M PB (pH 7.4), and air-dried overnight at 37°C. Sections were then defatted for 2 × 2 h in a mixture of chloroform/ethanol (1:1, vol.) and rinsed for 2 × 2 min in 100% ethanol, 1 × 2 min in 95% ethanol, and air-dried overnight at 37°C. Sections were then rehydrated through a graded series of ethanol solutions, 2 min in 95% ethanol, 2 min in 70% ethanol, 2 min in 50% ethanol; dipped in two separate baths of dH₂O; and stained for 20 s in a 0.25% thionin (Fisher Scientific, Waltham, MA; catalog No. T-409) solution, then dipped in two separate baths of dH₂O, 4 min in 50% ethanol, 4 min in 70% ethanol, 4 min in 95% ethanol + glacial acetic acid (1 drop per 100 mL of ethanol), 4 min in 95% ethanol, 2 × 4 min in 100% ethanol, 3 × 4 min in xylene; and coverslipped with DPX (BDH Laboratories, Poole, United Kingdom).

After brightfield, whole-slide imaging, we removed coverslips from NiDAB-stained sections by immersing them overnight in xylenes. After rehydration (1-min dips in a graded series of alcohols), we rinsed slides in ddH20 then dipped them in a fresh, filtered solution of 0.125% thionin (Fisher Scientific) for up to 60 s. Slides were then rinsed in ddH20 with tap water until the solution cleared and then dehydrated serially in 50% then 70% EtOH, fresh 95% EtOH (400 mL with ten drops of glacial acetic acid to clear excess thionin), 95%, 100%, and 100% EtOH, then two incubations in xylenes, from which we coverslipped each slide with Cytoseal.

After whole-slide imaging (described below), all slides from Syp-mCherry tracing cases were Nissl-counterstained and re-imaged. First, the coverslips were removed by soaking the slides in xylenes for 1–7 days. Then, after rehydration through 1-minute dips in a graded series of alcohols (400 mL each of 100%, 95%, 70%, 50% EtOH), we rinsed the slides in water and dipped them into a buffer solution containing 0.15% thionin (Fisher Scientific) for 1 minute. Slides were rinsed in tap water until the solution cleared, then dehydrated in an ascending series of alcohols for one minute each (50%, 70%, 95% with 10 drops of glacial acetic acid, 95%, 100%, and 100% EtOH), followed by two xylene solutions. Afterwards, the slides were coverslipped with Cytoseal.

The procedure for Nissl-stained sections followed our standard laboratory protocol described previously (Lavenex et al., 2009 (link)). Sections were taken out of the 10% formaldehyde solution, thoroughly washed 2 × 2 hours in 0.1 M phosphate buffer, mounted on gelatin-coated slides from filtered 0.05 M phosphate buffer (pH 7.4), and air-dried overnight at 37°C. Sections were then defatted 2 × 2 hours in a mixture of chloroform/ethanol (1:1, vol.), and rinsed 2 × 2 minutes in 100% ethanol, 2 minutes in 95% ethanol and air-dried overnight at 37°C. Sections were then rehydrated through a graded series of ethanol, 2 minutes in 95% ethanol, 2 minutes in 70% ethanol, 2 minutes in 50% ethanol, dipped in two separate baths of dH₂O, and stained 20 seconds in a 0.25% thionin (Fisher Scientific, Waltham, MA, cat# T-409) solution, dipped in 2 separate baths of dH₂O, 4 minutes in 50% ethanol, 4 minutes in 70% ethanol, 4 minutes in 95% ethanol + glacial acetic acid (1 drop per 100 ml of ethanol), 4 minutes in 95% ethanol, 2 × 4 minutes in 100% ethanol, 3 × 4 minutes in xylene, and coverslipped with DPX (BDH Laboratories, Poole, UK).