Anatomical Verification (Mendonça et al 2024)

Anaesthetize mice with isoflurane, followed by intraperitoneal injection of ketamine-xylazine (5 mg/kg xylazine; 100 mg/kg ketamine).

Perfuse transcardially in 1% phosphate buffered saline (PBS) and then 4% paraformaldehyde (PFA) in PBS.

Dissect brains after perfusion is completed.

Keep brains in 4% PFA solution overnight and then transferred to 1x PBS solution.

Section brains coronally in 30 to 50-um slices (using a Leica vibratome VT1000S) and keep in PBS before mounting or immunostaining.

Rinse 3 times with TBS (10 min each)

Peroxides treatment (inhibition peroxidase an permeabilization) 5 min

• 1.5 mL methanol
• 450 µL H₂O₂
• 13.05 mL TBS (this step is for DAB staining but we kept it because it favors the penetrance of the Ab)

Rinse 3 times with TBS (10 min each)

Membrane permeabilization treatment 15min (or 20min for Ab that don’t penetrate well)

• 300 µL Triton 10%
• 14.7 mL TBS

Rinse 3 times with TBS (10 min each)

Blockade with BSA 3% or other blocking solutions 1h (if needed)

Primary Antibody (diluted in TBS): overnight at 4°C (or longer period if needed)

TH (1:1000) Pel-Freez, Rogers, AR

Rinse 3 times with TBS (10 min each)

Secondary Antibody (diluted in TBS without) 40-45 min Room Temperature (1:400)

Rinse 2 times with TBS (10 min each) AND Rinse 2 times with TB (10 min each)

Mounting with Vectashield or PVA DABCO

Images were taken using a wide-field fluorescence microscope (Zeiss AxioImager).