Aug 22, 2023

Public workspaceMouse colon vasculature labeling combined with immunofluorescence

DOI
dx.doi.org/10.17504/protocols.io.e6nvwddd7lmk/v1
  • 1University of California, Los Angeles
  • Lixin Wang: We thank Ms. Honghui Liang (UCLA) for her excellent technical assistance.
Manmeet Bains
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DOI: dx.doi.org/10.17504/protocols.io.e6nvwddd7lmk/v1
Protocol Citation: Lixin Wang 2023. Mouse colon vasculature labeling combined with immunofluorescence. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwddd7lmk/v1
Manuscript citation:
Wang, L., P. Q. Yuan, C. Challis, S. Ravindra Kumar, and Y. Taché. 2022. "Transduction of Systemically Administered Adeno-Associated Virus in the Colonic Enteric Nervous System and c-Kit Cells of Adult Mice." Front Neuroanat 16: 884280. https://doi.org/10.3389/fnana.2022.884280.
Wang, L., P. Q. Yuan, and Y. Taché. 2023. "Vasculature in the mouse colon and spatial relationships with the enteric nervous system, glia, and immune cells." Front Neuroanat 17: 1130169. https://doi.org/10.3389/fnana.2023.1130169.
Yang, B., J. B. Treweek, R. P. Kulkarni, B. E. Deverman, C. K. Chen, E. Lubeck, S. Shah, L. Cai, and V. Gradinaru. 2014. "Single-cell phenotyping within transparent intact tissue through whole-body clearing." Cell158 (4): 945-958. https://doi.org/10.1016/j.cell.2014.07.017. https://www.ncbi.nlm.nih.gov/pubmed/25088144.
Yuan, P. Q., J. P. Bellier, T. Li, M. R. Kwaan, H. Kimura, and Y. Taché. 2021. "Intrinsic cholinergic innervation in the human sigmoid colon revealed using CLARITY, three-dimensional (3D) imaging, and a novel anti-human peripheral choline acetyltransferase (hpChAT) antiserum." Neurogastroenterol Motil 33 (4): e14030. https://doi.org/10.1111/nmo.14030.
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: August 16, 2023
Last Modified: August 22, 2023
Protocol Integer ID: 86562
Keywords: colon, fluorescent vessel painting, immunohistochemistry, microvessel, mouse, vasculature, wheat germ agglutinin
Funders Acknowledgement:
NIH
Grant ID: OT2OD024899
Abstract
To study the distribution, morphology and innervation of vasculature in different mouse colonic segments and layers, as well as spatial relationships of the vasculature with the enteric plexuses, glia and macrophages
Materials
Animals
Mice, C57BL/6J (000664, Jackson Laboratories, Sacramento, CA), male and female 8-12 weeks old

Reagents and supplies
  1. Isoflurane
  2. 0.9% saline
  3. wheat germ agglutinin-Alexa Fluor (WGA-AF) 448 (W11261, ThermoFisher Scientific)
  4. 0.01 M phosphate-buffer saline (PBS), stock solution 10X
  5. paraformaldehyde (P6148, Millipore-Sigma)
  6. Sylgard™ 184 silicone elastomer (Electron Microscopy Science, Hatfield, PA)
  7. Acrylamide
  8. VA-044 Initiator
  9. A4F0 hydrogel (Yang et al. 2014): 4% acrylamide and 0.25% VA-044 Initiator in 0.01M PBS. (A4F0: 4% acrylamide without paraformaldehyde)
  10. Sucrose
  11. Primary antibodies (Table 1)
  12. Secondary antibodies: donkey anti-rabbit IgG 555 or 647, donkey anti-rat IgG 594 (all purchased  from ThermoFisher)  
  13. Normal donkey serum
  14. Triton-X 100
  15. OCT Compound (Tissue-PlusTM, 23-730-571, Fisher Scientific)
  16. DAPI (4’, 6-diamidino-2-phenylindole; 62247, ThermoFisher Scientific)
  17. RIMS (refractive index matching solution) (Yang et al. 2014)
  18. Vectashield anti-fade mounting medium (Vector Laboratories, Burlingame, CA)
  19. iSpacer (Sunjin Lab, Hsinchu City, Taiwan, R.O.C.) (Yuan et al. 2021)
Table 1. Primary antibodies
ABCDEF
Antibody RRID* SpeciesSource Catalog No. Dilution
CD31 AB_393571 Rat BD Biosciences 550274 1:50
PGP9.5 AB_10891773 Rabbit Abcam ab108986 1:1,000
αCGRP AB_518147 Rabbit Peninsula T-4032 1:2,000
VIP AB_2890602 Rabbit CURE/UCLA ab7913 1:1,000
TH AB_390204 Rabbit Millipore AB152 1:1,000
GFAP AB_305808 Rabbit Abcam ab7260 1:2,000
S100B AB_882426 Rabbit Abcam AB56242 1:1,000
Iba1 AB_839504 Rabbit WAKO 019-19741 1:1,000
*: RRID: Research Resource Identifiers
Tissue collection and preparation
Tissue collection and preparation

Remove the whole colon is removed from the ileocecal junction to the end of distal colon at the level of pelvic brim where the iliac artery runs, after the cardiovascular perfusion of WGA.
Open the colon along the mesenteric margin, and pin the colon flat on a dish coated with Sylgard™ 184 silicone elastomer.
Fix the colon in 4% paraformaldehyde in 0.1M phosphate buffer (pH 7.4) overnight at 4°C.
Divide the colon samples to the proximal, mid and distal colon (Wang et al. 2022; Wang et al. 2023).
To improve penetration of antibodies for immunofluorescence, or for visualization of vasculature in different layers, the colonic samples could be prepared as the following approaches:
(1) Clear the samples using A4P0 hydrogel. The methodological details as described in previous report (Yang et al. 2014) and the protocol on protocols.io (Yuan et al. https://dx.doi.org/10.17504/protocols.io.bqi2muge).
(2) Treat the samples with 2% Triton-X 100 and 10% normal donkey serum in 0.01M PBS for 1 h at room temperature (RT) and then overnight at 4°C.
(3) Because the layers of the proximal colon with mucosal folds are thick and poorly immunolabeled, the proximal colon samples are prepared as: all layers without the mucosa and separating the wall in two parts at the submucosal layer.
(4) Frozen thick sections of colonic walls. The colon of mice perfused with the paraformaldehyde immersed in 20% sucrose for 2 days for cryoprotection, and embedded in Tissue-PlusTM O.C.T. Compound, snap-frozen and sectioned at 200 µm in a cryostat.
Vasculature painting
Vasculature painting

Dilute WGA AF-488 in 0.1M phosphate-buffer saline (PBS) at 30 µg/ml.
Anesthetized mice deeply with 5% isoflurane.
Flush the blood system with 0.9% saline via a cardiac cannula
Perfuse WGA-AF 488 (30 µg/g of body weight) at 1 ml/g of mouse body weight and a rate of 3 ml/min.

For immunostaining in thick colonic wall sections, some mice were perfused with 4% paraformaldehyde in 0.1M phosphate buffer (pH 7.4) following WGA perfusion.
Immunohistochemistry
Immunohistochemistry
Process samples of mice perfused with WGA-AF 488 by the following steps:
2% Triton-X 100 and 10% normal donkey serum in 0.01M PBS for 1 h at room temperature (RT) and then overnight at 4°C (the same step of 5(2) in Tissue Preparation)
Primary antibodies (Table 1) in 0.3% Triton-X 100-PBS for 2 h RT, at 4°C 2-5 days
Secondary antibodies for 3 h at RT, then overnight at 4°C.
Counterstaining: DAPI 1:2,000, 30 min at RT.
Mount the colonic tissues onto glass slides in a frame (iSpacer), and seal by glass coverslip in Vectashield anti-fade mounting medium or RIMS
Imaging and quantitative analysis
Imaging and quantitative analysis

Acquire microscopic images were acquired with Z-stacks in Zeiss confocal microscopes (LSM 710 and 880, Carl Zeiss Microscopy GmbH, Germany), using objectives of 10X, 20X and 63X.
The optical sections were scanned at intervals of 2 or 2.5 µm in 10X, 1 µm in 20X and 0.5 µm in 63X objectives.
The image segmentation, quantitation and visualization using Imaris 9.8 and 9.9 for neuroscientists (Bitplane Inc., Concord, MA).
The image segmentation, quantitation and visualization using Imaris 9.8 and 9.9 for neuroscientists (Bitplane Inc., Concord, MA).