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Whole-mount imaging of the mouse hindlimb vasculature using the lipophilic carbocyanine dye DiI.

ABSTRACT: The availability of transgenic disease backgrounds and the accessibility of molecular research reagents have contributed to make the mouse ischemic hindlimb the model of choice for many studies of angiogenesis, and to investigate new treatments for peripheral artery disease. A limitation of these models involves our inability to easily visualize the regenerated vascular architecture. Approaches such as micro-computed tomography and micro-angiography are expensive, technically demanding and not available to many laboratories. Here we describe a rapid and inexpensive adaptation of a vascular staining procedure for precise imaging of the mouse hindlimb vasculature. We introduced two technical modifications and an analytical extension to the original method including (i) pre-skinning of the muscle prior to fixation that preserves tissue integrity, (ii) mild pressure-desiccation subsequent to fixing that enhances resolution and image penetration, and (iii) reconstruction of confocal data into 3D images. The procedure provides resolution that is equivalent or superior to other approaches at a fraction of the cost, time and technology required.

SUBMITTER: Boden J

PROVIDER: S-EPMC4634701 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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Whole-mount imaging of the mouse hindlimb vasculature using the lipophilic carbocyanine dye DiI.

Boden Jeffrey J Wei Jianqin J McNamara George G Layman Hans H Abdulreda Midhat M Andreopoulos Fotios F Webster Keith A KA

BioTechniques 20120701 1

The availability of transgenic disease backgrounds and the accessibility of molecular research reagents have contributed to make the mouse ischemic hindlimb the model of choice for many studies of angiogenesis, and to investigate new treatments for peripheral artery disease. A limitation of these models involves our inability to easily visualize the regenerated vascular architecture. Approaches such as micro-computed tomography and micro-angiography are expensive, technically demanding and not a ...[more]

PMID: 26307258

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Project description:BackgroundThe applications of multiphoton microscopy for deep tissue imaging in basic and clinical research are ever increasing, supplementing confocal imaging of the surface layers of cells in tissue. However, imaging living tissue is made difficult by the light scattering properties of the tissue, and this is extraordinarily apparent in the mouse mammary gland which contains a stroma filled with fat cells surrounding the ductal epithelium. Whole mount mammary glands stained with Carmine Alum are easily archived for later reference and readily viewed using bright field microscopy to observe branching architecture of the ductal network. Here, we report on the advantages of multiphoton imaging of whole mount mammary glands. Chief among them is that optical sectioning of the terminal end bud (TEB) and ductal epithelium allows the appreciation of abnormalities in structure that are very difficult to ascertain using either bright field imaging of the stained gland or the conventional approach of hematoxylin and eosin staining of fixed and paraffin-embedded sections. A second advantage is the detail afforded by second harmonic generation (SHG) in which collagen fiber orientation and abundance can be observed.MethodsGFP-mouse mammary glands were imaged live or after whole mount preparation using a Zeiss LSM510/META/NLO multiphoton microscope with the purpose of obtaining high resolution images with 3D content, and evaluating any structural alterations induced by whole mount preparation. We describe a simple means for using a commercial confocal/ multiphoton microscope equipped with a Ti-Sapphire laser to simultaneously image Carmine Alum fluorescence and collagen fiber networks by SHG with laser excitation set to 860 nm. Identical terminal end buds (TEBs) were compared before and after fixation, staining, and whole mount preparation and structure of collagen networks and TEB morphologies were determined. Flexibility in excitation and emission filters was explored using the META detector for spectral emission scanning. Backward scattered or reflected SHG (SHG-B) was detected using a conventional confocal detector with maximum aperture and forward scattered or transmitted SHG (SHG-F) detected using a non-descanned detector.ResultsWe show here that the developing mammary gland is encased in a thin but dense layer of collagen fibers. Sparse collagen layers are also interspersed between stromal layers of fat cells surrounding TEBs. At the margins, TEBs approach the outer collagen layer but do not penetrate it. Abnormal mammary glands from an HAI-1 transgenic FVB mouse model were found to contain TEBs with abnormal pockets of cells forming extra lumens and zones of continuous lateral bud formation interspersed with sparse collagen fibers.ConclusionsCollagen fibril arrangement and TEB structure is well preserved during the whole mount procedure and light scattering is reduced dramatically by extracting fat resulting in improved 3D structure, particularly for SHG signals originating from collagen. In addition to providing a bright signal, Carmine Alum stained whole mount slides can be imaged retrospectively such as performed for the HAI-1 mouse gland revealing new aspects of abnormal TEB morphology. These studies demonstrated the intimate contact, but relatively sparse abundance of collagen fibrils adjacent to normal and abnormal TEBS in the developing mammary gland and the ability to obtain these high resolution details subject to the discussed limitations. Our studies demonstrated that the TEB architecture is essentially unchanged after processing.

Dataset Information

Whole-mount imaging of the mouse hindlimb vasculature using the lipophilic carbocyanine dye DiI.

Publications

Whole-mount imaging of the mouse hindlimb vasculature using the lipophilic carbocyanine dye DiI.

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