Three‐Dimensional Visualization of Blood and Lymphatic Vasculature in Tissue Whole Mounts Using Confocal Microscopy实验方法详情页

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Three‐Dimensional Visualization of Blood and Lymphatic Vasculature in Tissue Whole Mounts Using Confocal Microscopy

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Abstract
Table of Contents
Materials
Literature Cited

Abstract

Two vascular systems, cardiovascular and lymphatic, maintain appropriate interstitial and intravascular fluid volume in the body. Each is endowed with innate physiologic response capabilities activated upon tissue or organ ?damage.? Chronic activation following pathologic assault, however, can contribute to pathogenesis. Three?dimensional visualization of vasculature in whole tissues using confocal microscopy is a valuable tool for examining cellular and architectural changes accompanying altered vascular function. The relative affinities of plant lectins for carbohydrate moieties present on luminal surfaces of endothelial cells can be used to characterize endothelium in distinctive physiologic and pathologic states. Perivascular cells that wrap around blood endothelial cells can be visualized using antibodies immunoreactive with ??smooth muscle actin. Similarly, lymphatic endothelial cells can be detected by antibodies immunoreactive to the hyaluronan receptor LYVE?1. Together, these approaches allow functional and morphological analysis of blood vasculature distinct from endothelial cells within the lymphatic vascular network and surrounding support cells.

Keywords: blood vasculature; vascular leakage; lymphatics; whole mounts; fluorescence; confocal microscopy

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Basic Protocol 1: Direct Detection of Blood Vasculature in Tissue with Prefixation of Vasculature
Alternate Protocol 1: Direct Detection of Blood Vasculature in Tissue without Prefixation of Vasculature
Alternate Protocol 2: Direct Detection of Blood Vasculature and Exposed Regions of Subendothelial Basement Membrane
Alternate Protocol 3: Direct Detection of Blood and Lymphatic Vasculature in Tissue
Alternate Protocol 4: Direct Detection of Perivascular Support Cells in Blood Vasculature
Reagents and Solutions
Commentary
Literature Cited

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Basic Protocol 1: Direct Detection of Blood Vasculature in Tissue with Prefixation of Vasculature Materials

Mouse from which tissue is to be harvested
Anesthetic for inhalation: 2% (v/v) isoflurane in oxygen
1% (w/v) paraformaldehyde/0.5% (v/v) glutaraldehyde fixative solution (see recipe )
PBS ( appendix 2A )
PBS ( appendix 2A )/1.0% (w/v) BSA, filtered (0.44‐µm filter)
Fluorescein isothiocyanate (FITC)–conjugated Lycopersicon esculentum (tomato) lectin (Vector Laboratories; 2.0 mg/ml stock)
1% (w/v) paraformaldehyde fixative solution (see recipe )
Antifade mounting medium (e.g., Molecular Probes ProLong Gold antifade reagent)
Nitrocellulose‐based lacquer (nail polish) or rubber cement

Wide‐bore blunt needle (cannula) or blunted 16‐G needle connected to tubing
Hand‐held pump with attached manometer
Dissecting equipment, including dissecting microscope
24‐well tissue culture plates.
Glass microscope slides and coverslips
Laser scanning confocal microscope (Zeiss LSM 510 or a similar confocal system)
500‐to‐550‐nm band‐pass filter
Zeiss LSM Image Examiner or similar three‐dimensional analysis software

Additional reagents and equipment for administering inhalation anesthesia (Donovan and Brown, )

Alternate Protocol 1: Direct Detection of Blood Vasculature in Tissue without Prefixation of Vasculature

4% (w/v) paraformaldehyde fixative solution (see recipe )
0.5‐ml insulin syringe or 1.0‐ml syringe equipped with a 29‐G needle
60‐ml syringe equipped with a blunted 16‐G needle
Infusion pump for cardiac perfusion (KDS100; KD Scientific)

Alternate Protocol 2: Direct Detection of Blood Vasculature and Exposed Regions of Subendothelial Basement Membrane

Rhodamine‐conjugated R. communis (castor bean) agglutinin I (Vector Laboratories; 5.0 mg/ml stock)
4% (w/v) paraformaldehyde fixative solution (see recipe )
0.5‐ml insulin syringe or 1.0‐ml syringe equipped with a 29‐G needle
60‐ml syringe equipped with a blunted 16‐G needle

Infusion pump for cardiac perfusion (KDS100; KD Scientific)
565‐to‐615‐nm band‐pass filter or 650‐nm long‐pass filter

Alternate Protocol 3: Direct Detection of Blood and Lymphatic Vasculature in Tissue

4% (w/v) paraformaldehyde fixative solution (see recipe )
PBS ( appendix 2A )/0.3% (v/v) Triton X‐100
Normal goat serum
1:2000 rabbit anti‐mouse LYVE‐1 antibody (Upstate Biotechnology) in PBS ( appendix 2A )/0.3% (v/v) Triton X‐100
1:500 goat anti‐rabbit Alexa Fluor 594–conjugated secondary antibody or similar fluorochrome‐conjugated secondary antibody (Molecular Probes) in PBS ( appendix 2A )/0.3% (v/v) Triton X‐100
0.5‐ml insulin syringe or 1.0‐ml syringe equipped with a 29‐G needle
60‐ml syringe equipped with a blunted 16‐G needle

Infusion pump for cardiac perfusion (KDS100; KD Scientific)
Platform rocker
650‐nm long‐pass filter

Alternate Protocol 4: Direct Detection of Perivascular Support Cells in Blood Vasculature

Fluorescein isothiocyanate (FITC)–conjugated Lycopersicon esculentum (tomato) lectin (Vector Laboratories; 2.0 mg/ml stock) or FITC‐conjugated Ricinus communis (castor bean) agglutinin I (Vector Laboratories; 5.0 mg/ml stock)
4% (w/v) paraformaldehyde fixative solution (see recipe )
1:1000 Cy3‐conjugated anti–α‐smooth muscle actin mouse monoclonal antibody (Sigma) in PBS ( appendix 2A )/0.3% (v/v) Triton X‐100
Normal goat serum
PBS ( appendix 2A )/0.3% (v/v) Triton X‐100

0.5‐ml insulin syringe or 1.0‐ml syringe equipped with a 29‐G needle
60‐ml syringe equipped with a blunted 16‐G needle
Infusion pump for cardiac perfusion (KDS100; KD Scientific)
565‐to‐615‐nm band‐pass filter

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Literature Cited

Literature Cited
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