Slf0601

Formalin-fixed paraffin-embedded (FFPE) human tissues were received mounted on adhesive slides. The slides were baked overnight in an oven at 55°C (Robbin Scientific, Model 1000) and an additional 30 minutes at 65°C (Clinical Scientific Equipment, NO. 100). Tissues were deparaffinized with xylene and rehydrated with graded ethanol baths. Two step antigen retrieval was performed in the Decloaking Chamber (Biocare Medical) using the following settings: set point 1 (SP1), 125°C, 30 seconds; SP2: 90°C, 30 seconds; SP limit: 10°C. Slides were further incubated in hot Target Retrieval Solution, pH 9 (Agilent, S236784–2) for 15 minutes. Slides were then washed in two brief changes of diH2O (~2 seconds) and once for 5 minutes in 1x phosphate buffered saline (PBS), pH 7.4 (Fisher, BP39920). Sections were blocked in 10% normal goat serum (NGS, Vector S-1000), 1% bovine serum albumin (BSA, Sigma A7906) in PBS for 30 minutes at 20°C in a humid chamber, followed by PBS washes. Primary antibodies were diluted in 5% NGS, 1% BSA in 1x PBS and applied overnight at 4°C in a humid chamber, covered with plastic coverslips (Bio-Rad, SLF0601). Following overnight incubation, tissues were washed 3 × 10 min in 1x PBS. Coverslips (Corning; 2980–243 or 2980–245) were mounted in Slowfade Gold plus DAPI mounting media (Life Technologies, S36938).

After electrophysiology or imaging, slices with neurobiotin filled
axons were fixed overnight in 4% paraformaldehyde (PFA) in phosphate buffer
(PB, 0.1M, pH 7.6). Slices were subsequently stored in PB until
immunostaining and cleared using a modified CUBIC protocol, chosen because
it does not quench endogenous fluorescence (Susaki et al., 2015 (link)). For the immunostaining and CUBIC clearing,
all steps were performed at room temperature on a shaker plate. Slices were
placed in CUBIC reagent 1 for 1–2 days, washed in PB 3 × 1
hour each, placed in blocking solution (0.5% fish gelatin (Sigma) in PB) for
3 hours. Slices were directly placed in streptavidin-Cy5 conjugate at a
concentration of 1:1000 in PB for 2–3 days. Slices were washed 3
times for 2 hours each and were then placed in CUBIC reagent 2 overnight.
Slices were mounted on slides in reagent 2 in frame-seal incubation chambers
(Bio-Rad SLF0601) and coverslipped (#2 glass). Slices were imaged through
20×, 0.8 nA and 5×, 0.3 nA objectives on an LSM 800 confocal
microscope (Zeiss) and taken as tiled z-stacks using Zen Blue software in
the NINDS light imaging facility. Striatal axons were reconstructed using
Neurolucida (MBF bioscience).