Jul 16, 2023
Immunofluorescence staining of myenteric and submucosal plexuses
- 1Columbia University
Protocol Citation: Connor Monahan 2023. Immunofluorescence staining of myenteric and submucosal plexuses. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7p85kgwz/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: July 12, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 84907
Keywords: myenteric plexus, submucosal plexus, immunostain, immunofluorescence, enteric neuron, ileum, macrophage, T cell, ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: 0375
Abstract
This protocol details myenteric and submucosal plexus immunostaining.
Attachments
786-2005.pdf
53KB
Materials
Materials
Normal Donkey SerumJackson ImmunoResearch Laboratories, Inc.Catalog #017-000-121
Rabbit anti-tyrosine hydroxylase antibody; AB_390204Merck Millipore (EMD Millipore)Catalog #AB152
CD3 antibody | 145-2C11Bio-Rad LaboratoriesCatalog #MCA2690
IBA AbWakoCatalog #019-19741
Recombinant Anti-CD68 antibody [FA-11]AbcamCatalog #ab53444
- 1% Triton-X
- PBS
- DAPI
Procedure
Procedure
5h
5h
Note
Note: The plexuses are fragile. At each wash, use a micropipette and a dissecting microscope to carefully remove the solution from the well not suck up the plexus.
Cut out two small regions of each plexus sheet in PBS at 4 °C .
Add each tissue section to separate wells in a 96 well dish.
Add 10% normal donkey serum (Jackson Immunoresearch, Cat #017-000-121; West Grove, PA) and 1% Triton-X in PBS. Leave in blocking solution for 01:00:00 at Room temperature on a rotator.
1h
Incubate the tissue Overnight at Room temperature on a rotator with primary antibodies in 10% normal donkey serum, 1% Triton-X in PBS.
1h
Stain sections with antibodies against tyrosine hydroxylase (TH) (1:500, Millipore-Sigma, Cat # AB152; Burlington, MA) and CD3 (1:400, Bio-Rad Laboratories, Cat #MCA2690; Hercules, CA), ANNA1 (1:32,000; kind gift by the Gershon laboratory (Margolis et al., 2016)), Iba1 (1:500, WAKO, Cat # 019-19741; Richmond, VA), CD68 (1:1000, Abcam, Cat # ab53444; Waltham, MA).
On Day 2, remove the primary antibody solution from each well and wash.
Wash with PBS-Tween (0.1%) for 00:10:00 . (1/3)
10m
Wash with PBS-Tween (0.1%) for 00:10:00 . (2/3)
10m
Wash with PBS-Tween (0.1%) for 00:10:00 . (3/3)
10m
Incubate in secondary antibody (1:1000) in blocking solution for 02:00:00 at Room temperature , covered on a rotator.
2h
Wash again.
Wash for 00:10:00 each in 0.1% PBS-Tween. (1/3)
10m
Wash for 00:10:00 each in 0.1% PBS-Tween. (2/3)
10m
Wash for 00:10:00 each in 0.1% PBS-Tween. (3/3)
10m
Mount on slide with vectashield medium with DAPI
Imaging:
For imaging enteric neurons, for each plexus collect 2-3, 2x2 tile z-stack 640.17x640.17 µm confocal images at 20x magnification.
For imaging macrophages, for each plexus collect 2-3, 2x2 tile z-stack 390.09 x 390.09 µm confocal images at 20x magnification.
Analysis:
Count the number of ANNA1+, TH+ cells, and IBA1+ cells for each stacked image using Fiji.
Within the SP, threshold the TH+ signal, then analyze mean fluorescent intensity (MFI) and the area of the TH signal. Keep the thresholding consistent across each image, animal, and condition within each experiment.
Within each animal, sum the the number of ANNA1+ , TH+ cells, and IBA1+ cells across all images separately then divide by the acquisition area.
For each experiment, normalize to the CFA only condition.