Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology

Summary

Here, we present a protocol for the isolation of whole, intact mouse mammary glands to investigate extracellular matrix (ECM) expression and ductal morphology. Mouse #4 abdominal glands were extracted from 8-10 week old female nulliparous mice, fixed in neutral buffered formalin, sectioned and stained using immunohistochemistry for ECM proteins.

Abstract

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal architecture. Here, a small pocket below the skin was created using Mayo scissors, allowing separation of the glands within the subcutaneous tissue from the underlying peritoneum. Visualization of the glands was aided by the use of 3.5x-R surgical micro loupes. The pelt was inverted and pinned back allowing identification of the intact mammary fat pads. Each of the #4 abdominal glands was bluntly dissected by sliding the scalpel blade laterally between the subcutaneous layer and the glands. Immediately post-harvest, glands were placed in 10% neutral buffered formalin for subsequent tissue processing. Excision of the entire gland is advantageous because it primarily eliminates the risk of excluding important tissue-wide interactions between ductal epithelial cells and other microenvironmental cellular populations that could be missed in a partial biopsy. One drawback of the methodology is the use of serial sections from fixed tissues which limits analyses of ductal morphogenesis and protein expression to discrete locations within the gland. As such, changes in ductal architecture and protein expression in 3 dimensions (3D) is not readily obtainable. Overall, the technique is applicable to studies requiring whole intact murine mammary glands for downstream investigations such as developmental ductal morphogenesis or breast cancer.

Introduction

Breast cancer is characterized by a substantial degree of tissue fibrosis^1,2,3,4. Referred to as the ECM, this non-cellular entity is found in varying degrees in all tissues and is primarily comprised of a complex meshwork of fibrillar and non-fibrillar collagens, elastin, and glycoproteins in addition to various signaling molecules that are sequestered in this matrix. Under homeostatic conditions, the deposition and degradation of the ECM is tightly controlled.⁵ During breast tumorigenesis, the balance of ECM deposit....

Protocol

Procedures involving animal subjects in this protocol were reviewed and approved by the Institutional Animal Care and Use Committee of the Philadelphia College of Osteopathic Medicine and all techniques were conducted under strict ethical guidelines.

1. Sample Procurement and Processing

1. Select appropriate animal subject and place into a CO₂ chamber. For this experiment, use 8-10 week old female nulliparous B6.Cg-Cav1tmMIs/J and C57BI/6J.
2. Turn on gas flow to 30-40%. Once the animal is visibly unconscious after about 2 min, open the gas valve to full pressure for an additional 5 min.
   NOTE: Confirm anima....

Results

Female mice have 5 pairs of mammary glands. Specifically, there is one pair of cervical glands (#1), two pairs of thoracic glands (#2 and #3), one pair of abdominal glands (#4), and 1 pair of inguinal glands (#5) (Figure 1A). Here, we isolated the #4 glands as they are readily identifiable. In some circumstances, both #4 and #5 glands were isolated together as distinction between the two was difficult. To isolate intact #4 abdominal mammary g.......

Discussion

In the paper, we have described a technique to isolate intact mouse mammary glands for downstream histological analyses of ECM expression and ductal morphology. With respect to analyses of ductal morphology, this methodology enables the rapid investigation of ductal architecture based off of stained histological sections. Other methods of ductal analyses rely on injections of dyes to enable visualization of the ductal tree, methods which may be technically challenging and time consuming.

In br.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Light Microscope	Olympus	BX43
Microscope Camera	Olympus	DP73
Image Analysis Software	Olympus	cellSens Entry software
	NIH	ImageJ
3.5x-R Surgical Micro Loupes	Rose Micro Solutions		Magnification at researcher's preference
Mayo Scissors	Medline	DYND04035
Staining Rack	Fisher Scientific	121
Staining Dish	Fisher Scientific	112
Coplin Jars	Fisher Scientific	19-4
Glass coverslips	Fisher Scientific	12-550-15	Size appropriate for tissue
IHC EnVision+ Kit (HRP, Mouse, DAB+)	Dako	K400611-2
Picrosirius Red Kit	Abcam	AB150681
Eosin Y, alcoholic	Sigma-Aldrich	HT110132
Harris Hematoxylin	Sigma-Aldrich	HHS16
Donkey Serum	EMD Millipore	S30
10% Neutral Buffered Formalin	Sigma-Aldrich	HT501128
Xylenes, Reagent Grade	Sigma-Aldrich	214736
Ethanol, 200 proof	Sigma-Aldrich	792780	suitable for molecular biology
Phosphate Buffered Saline, 1x	Gibco	10010023
Sodium Citrate	Fisher Scientific	S279-500
Calcium Carbonate	Sigma-Aldrich	202932
Permanent Mounting Medium	Dako	S1964
Eukitt's Mounting Medium	Sigma-Aldrich	3989
Fibronectin antibody	Abcam	AB23750
Tenascin-C antibody	Abcam	AB108930
Alpha Smooth Muscle Actin antibody	Abcam	AB124964
Dako Envision Dual Link System HRP	Dako	K4065