Jun 03, 2024

Public workspaceImmunofluorescence labelling and imaging of cholinergic interneurons in post-mortem human brain tissues

DOI
dx.doi.org/10.17504/protocols.io.eq2lywn5qvx9/v1
  • Alan K. L. Liu1,2,
  • Laura Parkkinen1,2
  • 1Oxford Parkinson’s Disease Centre, University of Oxford, Oxford OX1 3PT, UK;
  • 2Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford OX3 9DU, UK
Cláudia C. Mendes
Open access
DOI: dx.doi.org/10.17504/protocols.io.eq2lywn5qvx9/v1
Protocol Citation: Alan K. L. Liu, Laura Parkkinen 2024. Immunofluorescence labelling and imaging of cholinergic interneurons in post-mortem human brain tissues. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lywn5qvx9/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: May 09, 2024
Last Modified: June 03, 2024
Protocol Integer ID: 99495
Abstract
This protocol details immunofluorescence labelling and imaging of cholinergic interneurons along with striatal astrocytes in formalin-fixed paraffin-embedded (FFPE) post-mortem human brain tissues.
Collection and fixation of post-mortem human brain tissues
Collection and fixation of post-mortem human brain tissues
Collect 6 µm-thick sections of formalin-fixed paraffin-embedded (FFPE) tissues containing the anterior basal ganglia at the level of nucleus accumbens.
Place tissue sections in an oven at 70°C for 30 mins.
Dewax tissue sections in xylene (3 x 5 mins).
Rehydrate tissue sections through decreasing concentration of industrial denatured alcohol (IDA) (100%, 100%, 90%, 70%; 5 mins each)and subsequently in distilled water (5 mins).
Primary Antibody
Primary Antibody
Perform heat-induced epitope retrieval by placing tissue sections in autoclave (121°C, 20 mins) in 0.01 M sodium citrate buffer (pH 6.0).
Rinse in PBS (3 x 5 mins).
Incubate samples at 4°C overnight with a mixture of the following primary antibodies:
  • anti-ChAT antibodies (1:50, AB144P; Millipore, UK; RRID:AB_2313845)
  • anti-GFAP antibodies (1:2000; Z0334; Agilent Dako, Santa Clara, United States; RRID:AB_10013382).

Note
Antibodies were diluted in 0.3% Triton X-100, 2% fetal bovine serum (FBS) and PBS.

Secondary Antibody
Secondary Antibody
Rinse samples in PBS (3 x 5 mins).
Prepare Secondary Antibody mixture of Alexa Fluor 488-conjugated donkey anti-rabbit secondary antibody (1:200; A-11055; ThermoFisher Scientific, UK) and Alexa Fluor 594-conjugated donkey anti-goat secondary antibody (1:200; ThermoFisher Scientific, UK) diluted in 0.1% Triton-X in PBS.
Incubate sections in Secondary Antibody mixture in the dark (covered with foil) for 1 hour at room temperature.
Mounting
Mounting
Incubate sections with TrueBlack® Lipofuscin Autofluorescence Quencher (1:20 with 70% ethanol; Biotium, Fremont, CA, United States) for 30 seconds to block endogenous fluorescence signal.
Rinse in PBS (3 x 2 mins).
Mount and coverslip with Vectashield antifade mounting medium with DAPI (H1900, Vector Laboratories, Peterborough, UK).
Confocal Imaging and Analysis
Confocal Imaging and Analysis
Visualise immunofluorescent-stained slides using Zeiss 880 Airyscan inverted confocal microscope (Carl Zeiss).
Acquire images using 10x objectives (Plan-Apochromat/0.45 NA) and 63x objectives (Plan-Apochromat, oil immersion; DIC M27/1.4 NA) with laser excitation at 405 nm (solid state), 488 nm (Argon), and 561 nm (solid state).
Perform image capture and processing using the Zen Black and Zen Blue (Carl Zeiss,Germany) software.