Methods for preparing fluorescent and neutral red-stained whole mounts of mouse mammary glands - PubMed (original) (raw)

Methods for preparing fluorescent and neutral red-stained whole mounts of mouse mammary glands

John D Landua et al. J Mammary Gland Biol Neoplasia. 2009 Dec.

Abstract

Whole mount preparations of mouse mammary glands are useful for evaluating overall changes in growth and morphology, and are essential for detecting and evaluating focal or regionally-localized phenotypes that would be difficult to detect or analyze using other techniques. We present three newly developed methods for preparing whole mounts of mammary glands from genetically-engineered mice expressing fluorescent proteins, as well as using either neutral red or a variety of fluorescent dyes. Unlike traditional hematoxylin- or carmine-stained preparations, neutral red-stained and some fluorescent preparations can be used for several common downstream analyses.

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Figures

Figure 1

Figure 1

Whole mounts of mouse mammary glands expressing fluorescent proteins. A. Mature thoracic (#3) mammary gland harvested from a mouse expressing the ECFP gene driven by the cytomegalovirus (CMV) immediate early enhancer coupled to the chicken beta actin promoter. Bar represents 1mm. B. ECFP gland shown at higher magnification. C. Thoracic (#3) mammary gland harvested from a mature mouse expressing the EGFP gene driven by the CMV immediate early enhancer coupled to the chicken beta actin promoter. D. EGFP-expressinggland shown at higher magnification. E. Thoracic (#3) mammary gland harvested from a mature mouse expressing a membrane-targeted tdTomato driven by the chicken beta actin promoter. F. tdTomato-expressing mammary gland shown at higher magnification. G. Whole mount of an ECFP expressing mammary gland imaged using confocal microscopy. H. Mammary duct from a mouse expressing a fibroblast specific protein-1 (Fsp-1) Cre recombinase and a Cre reporter which expresses the tdTomato protein in non-recombined cells and EGFP in recombined cells that was imaged using confocal microscopy. Fibroblasts within the stroma appear green indicating active expression of Cre recombinase while the ductal epithelium remains red indicating a lack of Cre expression.

Figure 2

Figure 2

Whole mount preparations of mouse mammary glands stained with fluorescent dyes. A. Tthoracic (#3) mammary gland from a mature mouse stained with ethidium bromide. Bar represents 1mm. B. Ethidium bromide stained gland shown at higher magnification. C. Mature #3 thoracic mammary gland stained with propidium iodide. D. Propidium iodide stained gland shown at higher magnification. E. Tthoracic (#3) mammary gland from a mature mouse stained with DAPI. B. DAPI-stained mammary gland shown at higher magnification.

Figure 3

Figure 3

Whole mount preparation of a mouse mammary gland stained with neutral red and subsequent immunohistochemistry. A. Thoracic (#3) mammary gland harvested from a mouse at 9-weeks-of-age stained with neutral red. Bar represents 1mm. B. Neutral red stained gland shown at higher magnification. C. Immunohistochemistry using a monoclonal antibody for 5-bromo-2-deoxyuridine (Brdu)conducted after neutral red staining Bar represents 20µm. D. Immunohistochemistry using a polyclonal antibody against GFP conducted after neutral red staining. Mouse was genetically modified to express EGFP.

References

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