TITLE: Mouse colon vasculature labeling combined with immunofluorescence
AUTHORS: Lixin Wang

[DESCRIPTION]
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




[STEPS]
SECTION: Tissue collection and preparation
1. 
SECTION: Vasculature painting
2. 
SECTION: Vasculature painting
2.1. Dilute WGA AF-488 in 0.1M phosphate-buffer saline (PBS) at 30 µg/ml.
SECTION: Vasculature painting
2.2. Anesthetized mice deeply with 5% isoflurane.
SECTION: Tissue collection and preparation
1.1. 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.
SECTION: Vasculature painting
2.3. Flush the blood system with 0.9% saline via a cardiac cannula
SECTION: Vasculature painting
2.4. 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.
SECTION: Tissue collection and preparation
1.2. Open the colon along the mesenteric margin, and pin the colon flat on a dish coated with Sylgard™ 184 silicone elastomer.

SECTION: Tissue collection and preparation
1.3. Fix the colon in 4% paraformaldehyde in 0.1M phosphate buffer (pH 7.4) overnight at 4°C.

SECTION: Tissue collection and preparation
1.4. Divide the colon samples to the proximal, mid and distal colon (Wang et al. 2022; Wang et al. 2023).
SECTION: Tissue collection and preparation
1.5. 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.
SECTION: Immunohistochemistry
3. Process samples of mice perfused with WGA-AF 488 by the following steps:
SECTION: Immunohistochemistry
3.1. 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)
SECTION: Immunohistochemistry
3.2. Primary antibodies (Table 1) in 0.3% Triton-X 100-PBS for 2 h RT, at 4°C 2-5 days
SECTION: Immunohistochemistry
3.3. Secondary antibodies for 3 h at RT, then overnight at 4°C.
SECTION: Immunohistochemistry
3.4. Counterstaining: DAPI 1:2,000, 30 min at RT.
SECTION: Immunohistochemistry
3.5. Mount the colonic tissues onto glass slides in a frame (iSpacer), and seal by glass coverslip in Vectashield anti-fade mounting medium or RIMS
SECTION: Imaging and quantitative analysis
4. 
SECTION: Imaging and quantitative analysis
4.1. 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.
SECTION: Imaging and quantitative analysis
4.2. 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.
SECTION: Imaging and quantitative analysis
4.3. The image segmentation, quantitation and visualization using Imaris 9.8 and 9.9 for neuroscientists (Bitplane Inc., Concord, MA).
SECTION: Imaging and quantitative analysis
4.4. The image segmentation, quantitation and visualization using Imaris 9.8 and 9.9 for neuroscientists (Bitplane Inc., Concord, MA).