Jan 16, 2024
Anatomical Verification (Mendonça et al 2024)
- Marcelo D Mendonça1,2,3
- 1Champalimaud Research, Champalimaud Foundation, Lisbon 1400-038, Portugal;
- 2Champalimaud Clinical Centre, Champalimaud Foundation, Lisbon 1400-038, Portugal;
- 3NOVA Medical School | Faculdade de Ciências Médicas, Universidade Nova de Lisboa, Lisbon 1169-056, Portugal
Protocol Citation: Marcelo D Mendonça 2024. Anatomical Verification (Mendonça et al 2024). protocols.io https://dx.doi.org/10.17504/protocols.io.14egn3o3ql5d/v1
Manuscript citation:
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 12, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 93475
Keywords: ASAPCRN
Abstract
Protocol for anatomical verification (sample prep, immunostaining and imaging) in Mendonça et al 2024.
Materials
Standard perfusion setup
Vibrotome
Before start
Animals were euthanized after completion of the behavioural tests.
Sample Preparation
Sample Preparation
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.
Immunostaining for TH (Day 1)
Immunostaining for TH (Day 1)
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
Immunostaining for TH & Mounting (Day 2)
Immunostaining for TH & Mounting (Day 2)
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
Imaging
Imaging
Images were taken using a wide-field fluorescence microscope (Zeiss AxioImager).