Jul 12, 2022

Public workspacePost mortem human substantial nigra TH staining

DOI
dx.doi.org/10.17504/protocols.io.n2bvj68d5lk5/v1
  • Waijiao Kai1,
  • Xiqun Chen2
  • 1MassGeneral Institute for Neurodegenerative Disease, Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, USA Department of Integrative Medicine, HuaShan Hospital, Institutes of Integrative Medicine, Fudan University, Shanghai, China;
  • 2MassGeneral Institute for Neurodegenerative Disease, Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, USA Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, Towson, MD, USA
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DOI: dx.doi.org/10.17504/protocols.io.n2bvj68d5lk5/v1
Protocol Citation: Waijiao Kai, Xiqun Chen 2022. Post mortem human substantial nigra TH staining. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj68d5lk5/v1
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: February 18, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 58415
Keywords: ASAPCRN
Abstract
This protocol is standardized for postmortem (frozen) SN tissue from pathologically diagnosed PD patients and control individuals for Immunofluorescence staining.
Materials
Anti-TH, Sigma T1299, 1:400
Secondary antibody - goat anti-mouse Alexaflour 488, Lifetech, Cat# A11001) 1:400
ProLong Gold antifade (Lifetech, P36935)
Deparaffinization
Wash slides in 2 changes of Amount300 mL ml of xylene – Duration00:06:00 each at room temperature.

6m
Rehydration
Wash slides in Amount300 mL of 100% alcohol –Duration00:09:00 at TemperatureRoom temperature .
Wash slides inAmount300 mL of 95% alcohol – Duration00:06:00 at TemperatureRoom temperature .
Wash slides in Amount300 mL of 80% alcohol – Duration00:03:00 at TemperatureRoom temperature .
Rinse slides in gentle running distilled water – Duration00:05:00 atTemperatureRoom temperature .

23m
Antigen retrieval
Microwave (high power) slides in Amount200 mL Concentration0.01 Molarity (M) sodium citrate buffer, pH 6.0 at Temperature99-100 °C for Duration00:05:00 , three rounds.
Add Amount40 mL distill water after 1st round
Add Amount150 mL sodium citrate buffer after the 2nd round.
Let stand at room temperature in the buffer - Duration00:20:00
Rinse in 1X TBS with Tween (TBST) – for Duration00:05:00 at TemperatureRoom temperature .

30m
Immunostaining
Do not allow tissues to dry at any time during the staining procedure.
Apply a universal protein block (5% goat serum in TBST) – for Duration00:30:00 at TemperatureRoom temperature .
Drain protein block from slides, and apply diluted primary antibody overnight at Temperature4 °C .
Rinse slides in 1X TBST 3 times - Duration00:05:00 each at TemperatureRoom temperature .
Incubate secondary antibody (Conc - 1:400) - Duration00:30:00 at Temperature37 °C .
Rinse slides 1X TBST 3 times – Duration00:05:00 each at TemperatureRoom temperature
Mount the slides with ProLong Gold antifade
1h 10m