Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy

Summary

We describe a protocol to surgically expose and stabilize the murine submandibular salivary gland for intravital imaging using upright intravital microscopy. This protocol is easily adaptable to other exocrine glands of the head and neck region of mice and other small rodents.

Abstract

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, including cell biology, oncology, dentistry, and immunology. The SMG is an exocrine gland comprised of secretory epithelial cells, myofibroblasts, endothelial cells, nerves, and extracellular matrix. Dynamic cellular processes in the rat and mouse SMG have previously been imaged, mostly using inverted multi-photon microscope systems. Here, we describe a straightforward protocol for the surgical preparation and stabilization of the murine SMG in anesthetized mice for in vivo imaging with upright multi-photon microscope systems. We present representative intravital image sets of endogenous and adoptively transferred fluorescent cells, including the labeling of blood vessels or salivary ducts and second harmonic generation to visualize fibrillar collagen. In sum, our protocol allows for surgical preparation of mouse salivary glands in upright microscopy systems, which are commonly used for intravital imaging in the field of immunology.

Introduction

Saliva is secreted by exocrine glands to lubricate food, protect the mucosal surfaces of the oral tract, and to deliver digestive enzymes as well as antimicrobial substances^1,2. In addition to minor salivary glands interspersed in the oral submucosa, there are three bilateral sets of major glands identified as parotid, sublingual, and submandibular, according to their location^1,2. Pyramid-shaped epithelial cells, organized into flask-shaped sacs (acini) or demilunes that are surrounded by myoepithelial cells and a basement membrane, secrete the serous ....

Protocol

All animal work has been approved by the Cantonal Committee for Animal Experimentation and conducted according to federal guidelines.

1. Anesthetize the Mouse

1. Wear personal protective equipment, including lab coat and gloves.
2. Mix ketamine, xylazine, and saline to a working concentration of 20 mg/mL and 1 mg/mL, respectively. Inject the working stock intraperitoneally (i.p.) at 8-10 µL/g of mouse. Place the mouse back into the cage.
   NOTE: This protocol has been tested for 6- to 40-week-old male and female mice with a C57BL/6 background.
3. Prepare the acepromazine solution at 2.5 mg/mL and inject 30 &....

Results

This protocol allows imaging of almost the entire dorsal or ventral side of the SMG. The field of view typically also includes the sublingual salivary gland, which differs slightly from the SMG in cellular composition⁴. Both glands are encapsulated by fibrillar collagen and subdivided into lobes. Most 2PM systems can produce a label-free image of fibrillar collagen by measuring the 2^nd harmonic signal, but usually fluorescent molecules are needed for vis.......

Discussion

This protocol offers a straightforward approach for in vivo imaging of murine submandibular and sublingual salivary glands using upright non-linear microscopy often used in the field of immunology. The method can be adapted for the imaging of other exocrine glands in the head and neck region. For instance, our lab has performed imaging of the lacrimal gland in an analogous manner (not shown).

Removing the connective tissue around the SMG is the most critical step of this protocol, sin.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Narketan 10 %  (Ketamine) 20ml (100 mg/ml)	Vetoquinol	3605877535982
Rompun 2% (Xylazine) 25 ml (20 mg/ml)	Bayer	680538150144
Saline NaCl 0.9%	B. Braun	3535789
Prequillan 1% (Acepromazine) 10 ml (10 mg/ml)	Fatro	6805671900029
Electric shaver	Wahl	9818L	or similar
Hair removal cream	Veet	4002448090656
Durapore Surgical tape (2.5 cm x 9.1 m and 1.25 cm x 9.1 m)	Durapore (3M)	1538-1
Durapore Surgical tape (2.5 cm x 9.1 m and 1.25 cm x 9.1 m)	Durapore (3M)	1538-0
Super glue Ultra gel, instantaneous glue	Pattex, Henkel	4015000415040
Microscope cover glass slides 20 mm and 22 mm	Menzel-Gläser	631-1343/ 631-1344
Grease for laboratories 60 g glisseal N	Borer (VWR supplier)	DECO514215.00-CA15
Surgical scissors	Fine Science Tools (F.S.T )	14090-09	or similar
Fine Forceps	Fine Science Tools (F.S.T )	11252-20	or similar
Cotton swab	Migros	617027988254	or similar
Gauze Gazin 5 x 5 cm	Lohmann and Rauscher	18500	or similar
Stereomicroscope	Leica	MZ16	or similar
Texas Red dextran 70kDa	Molecular Probes	D1864
Cascade Blue dextran 10kDa	invitrogen	D1976
Two-photon system	LaVision Biotec	TrimScope I and II	or similar
XLUMPLANFL 20x/0.95 W objective	Olympus	n/a	or other water immersion objective
Digital thermometer	Fluke	95969077651