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Abstract

Introduction

Protocol

Representative Results

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Materials

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Biology

Modified In Vivo Matrix Gel Plug Assay for Angiogenesis Studies

Published: June 30th, 2023

DOI:

10.3791/65567

1Pingyang Affiliated Hospital of Wenzhou Medical University, 2School of Pharmaceutical Sciences, Wenzhou Medical University
* These authors contributed equally

The method presented here can evaluate the effect of reagents on angiogenesis or vascular permeability in vivo without staining. The method uses dextran-FITC injection via the tail vein to visualize neo-vessels or vascular leakage.

Several models have been developed to investigate angiogenesis in vivo. However, most of these models are complex and expensive, require specialized equipment, or are hard to perform for subsequent quantitative analysis. Here we present a modified matrix gel plug assay to evaluate angiogenesis in vivo. In this protocol, vascular cells were mixed with matrix gel in the presence or absence of pro-angiogenic or anti-angiogenic reagents, and then subcutaneously injected into the back of recipient mice. After 7 days, phosphate buffer saline containing dextran-FITC is injected via the tail vein and circulated in vessels for 30 min. Matrix gel plugs are collected and embedded with tissue embedding gel, then 12 µm sections are cut for fluorescence detection without staining. In this assay, dextran-FITC with high molecular weight (~150,000 Da) can be used to indicate functional vessels for detecting their length, while dextran-FITC with low molecular weight (~4,400 Da) can be used to indicate the permeability of neo-vessels. In conclusion, this protocol can provide a reliable and convenient method for the quantitative study of angiogenesis in vivo.

Angiogenesis, the process of formation of neo-vessels from pre-existing vessels, plays a critical role in many physiological and pathological processes, such as embryonic development, wound healing, atherosclerosis, tumor development, etc.1,2,3,4,5. This dynamic process involves several steps, including the degradation of the matrix, vascular cell proliferation, migration and self-organization to form tubular structures and the stabilization of the neo-vessels6. Promoting angiogenesi....

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All procedures involving animal subjects were approved by the Institutional Animal Care and Use Committee (IACUC) of Wenzhou Medical University (XMSQ2021-0057, July 19th, 2021). All reagents and consumables are listed in the Table of Materials.

1. Culture medium preparation

  1. 10x M199 culture medium: Dissolve M199 powder to 10x concentration with 90 mL of deionized water and add 10 mL of fetal bovine serum (FBS), then pass through a 0.22 &.......

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Figure 1 is the flowchart depicting how to prepare the mixture of matrix gel, vascular cells, culture medium and reagent. The mixture was then subcutaneously injected into the back of Nu/Nu mice and heated using a heating pad to accelerate its coagulation to finally form gel plug.

Figure 2A is the flowchart to indicate vessels with fluorescent labeled dextran. Fluorescent labeled dextran was injected via the tail vein and circle for 3.......

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We present a reliable and convenient method for the quantitative evaluation of angiogenesis in vivo without staining. In this protocol, vascular cells were mixed with matrix gel in the presence of pro-angiogenic or anti-angiogenic reagents, and then subcutaneously injected into the back of Nu/Nu mice to form gel plug (Figure 1). After 7 days of gel plug formation, dextran-FITC was intravenously injected and circulated for 30 min. The gel plug was collected and embedded with tissue e.......

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This work was funded by Natural Science Foundation of Zhejiang Province (LY22H020005), and National Natural Science Foundation of China (81873466).

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NameCompanyCatalog NumberComments
Adhesion Microscope SlidesCITOTEST188105
Anesthesia SystemRWDR640-S1
Cell CounterInvitrogenAMQAX1000
Cell Culture DishCorning430167
CryoslicerThermo FisherCryoStar NX50
Dextrans-FITC-150kDaWEIHUA BIOWH007N07
Dextrans-FITC-4kDaWEIHUA BIOWH007N0705
Embedding CassettesCITOTEST80203-0007
Endothelial Cell MediumScienCell35809
Endothelial Growth SupplementsScienCell1025
Fetal Bovine SerumGibco10100147C
Fibroblast Growth Factor 1AtaGenix9043p-082318-A01FGF1
Fluorescence MicroscopeNikonECLIPSE Ni
Heating PadBoruida30-50-30
Insulin SyringeBD300841
IsofluraneRWDR510-22-10
Laboratory BalanceSartoriusBSA124S-CW
MatrigelCorning356234Matrix gel
Medium 199 powderGibco31100-035
MicrotubesAxygenMCT-150-C
Optimal Cutting Temperature (OCT) CompoundSUKURA4583Tissue embedding gel
Palmitate AcidKunChuangKC001
Penicillin-Streptomycin LiquidSolarbioP1400
Phosphate Buffer SalineSolarbioP1022
Surgical InstrumentsRWDRWD
Tail Vein Injection InstrumentKEW BASISKW-XXY
Trypsin-EDTA SolutionSolarbioT1320
Ultra-Low Temperature FreezereppendorfU410
Vascular Endothelial Growth FactorCHAMOTCM058-5HPVEGF

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