体内哌莫硝唑缺氧研究

Kristina  Y.  Aguilera; Rolf  A.  Brekken

doi:10.21769/BioProtoc.1254

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Peer-reviewed

Hypoxia Studies with Pimonidazole in vivo

体内哌莫硝唑缺氧研究

Kristina Y. Aguilera

Rolf A. Brekken email

发布: 2014年10月05日第4卷第19期 DOI: 10.21769/BioProtoc.1254 浏览次数: 27259

评审: Ralph BottcherHongLok Lung

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参见作者原研究论文

The authors used this protocol in:

Cover of Cancer Research, featuring study using the protocol.

Feb 2014

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Abstract

Therapy-induced hypoxia drives changes in the tumor microenvironment that contribute to the poor response to therapy. Hypoxia is capable of driving the expression and/or activation of specific signaling cascades (e.g., c-Met, Axl, CTGF), the recruitment of tumor promoting immune cells, and the induction of cell survival pathways including autophagy (Phan et al., 2013; Hu et al., 2012; Ye et al., 2010). We have recently shown that anti-VEGF therapy-induced hypoxia can result in changes in the extracellular matrix that contribute to the aggressiveness of tumors post therapy (Aguilera et al., 2014). Importantly, therapies that induce hypoxia do not always increase epithelial plasticity and tumor aggressiveness (Ostapoff et al., 2013; Cenik et al., 2013). We have used pimonidazole to evaluate hypoxia in tumors and herein provide a detailed protocol for this useful tool to interrogate the levels of hypoxia in vivo.

The utility of the Hypoxyprobe^TM (pimonidazole hydrochloride) immunohistochemical analysis approach allows for the assessment of hypoxia in different tissues as well as cell types. Pimonidazole is a 2-nitroimidazole that is reductively activated specifically in hypoxic cells and forms stable adducts with thiol groups in proteins, peptides, and amino acids (Cenik et al., 2013; Arnold et al., 2010; Raleigh and Koch, 1990; Raleigh et al., 1998). Furthermore, the amount of pimonidazole that is detected is directly proportional to the level of hypoxia within tumors.

Materials and Reagents

Hypoxyprobe^TM-1 Plus Kit (Hypoxyprobe^TM, catalog number: HP1-1000Kit )
A rabbit, FITC-conjugated, secondary reagent is included in the Hypoxyprobe^TM-1 Plus Kit (Hypoxyprobe^TM, catalog number: HP1-1000Kit)
Sodium chloride ACS reagent (Sigma-Aldrich, catalog number: S9888 )
Milli Q H₂O
Corning bottle-top vacuum filter system (Sigma-Aldrich, catalog number: CLS430769 )
PBS (Sigma-Aldrich, catalog number: P-4417 )
Anti-CD31 antibody (Dianova GmbH, catalog number: DIA-310 )
Anti-Meca32 antibody (Abcam, catalog number: ab27853 )
Anti-Endomucin antibody (Millipore, catalog number: MAB2624 )
Cy3-conjugated secondary IgG (Jackson ImmunoResearch Laboratories)
Tissue-Tek OCT compound (Ted Pella, catalog number: 27050 )
Liquid nitrogen
IsoFlo (isoflurane, USP) (Abbott Laboratories, catalog number: 05260-05 )
Carbon dioxide (Sigma-Aldrich, catalog number: 295108 -227G)
Superfrost plus; white microscope slides (Thermo Fisher Scientific, catalog number: 12-550-15 )
Liquid scintillation vials, plastic (Sigma-Aldrich, catalog number: M2026 -1000EA)
Prolong gold antifade mountant (Life Technologies, catalog number: P10144 )
Aqua Block/EIA/WB (EastCoast Bio, catalog number: PP82 )
Acetone (Sigma-Aldrich, catalog number: 320110 )
Micro cover glasses, rectangular, No. 1 (VWR International, catalog number: 48393-106 )
Tween 20 (Sigma-Aldrich, catalog number: P9416 -50ML)
0.9% saline (see Recipes)
30 mg/ml pimonidazole in 0.9% saline (see Recipes)
Antibody diluent (see Recipes)
20% aqua block (see Recipes)
PBS-t (see Recipes)

Equipment

Cryostat (Leica, catalog number: CM3050 S )
Low profile microtome blades (Accu Edge, catalog number: 4689 )
Surgical equipment (Braintree Scientific)
Fluorescent or bright-field microscope (Nikon Eclipse, model: E600 )
Fluorescent microscope camera (Photometrics, Cool Snap HQ)
Nikon filters (FITC: 96170M F HQ, Cy3/TRITC: 96171M R HG, DAPI/nuclei: 96101M UV-2E/C)
30 G x 1/2 in. precision glide needles (BD Biosciences, catalog number: 305106 )
Tuberculin syringe only (BD Biosciences, catalog number: 309659 )
Tailveiner restrainer for mice (Braintree Scientific, catalog number: TV-150 STD )