Intravital Visualization of Gamma Delta Intraepithelial Lymphocytes

Abstract

Source: Jia, L., et al. Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine. J. Vis. Exp. (2019)

This video demonstrates the visualization of γδ intraepithelial lymphocytes (IELs) in transgenic mice using confocal microscopy. The surgical procedure involves creating a perforation line and making a longitudinal incision to expose the intestinal mucosa and villi. The transgenic mice express reporter proteins exclusively in γδ IELs, allowing their tracking. Intravital imaging captures the dynamic movement of green fluorescence-labeled γδ IELs along the intestinal epithelial cell's basement membrane.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Mouse Preparation

NOTE: The following procedure, including animal preparation and surgery, will take 30–40 min. Prior to the surgery, turn on the microscope and warm up the enclosed incubator on the microscope to 37 °C.

1. Perform experiments on 8–10 weeks old TcrdEGFP mice on a C57BL/6 background, which were obtained from Bernard Malissen (INSERM, Paris, France). According to the IACUC-approved procedure, an....

Results

Figure 1: Using forceps to create a hole in the mesentery. Place forceps on either side of the membrane to create a hole for the sutures.

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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
35mm dish, No. 1.5 Coverslip	MatTek	P35G-1.5-14-C
Alexa Fluor 633 Hydrazide	Invitrogen	A30634
BD PrecisionGlide Hypodermic needles - 27g	Thermo Fisher Scientific	14-826-48
BD Slip Tip Sterile Syringe - 1 ml	Thermo Fisher Scientific	14-823-434
BD Tuberculin Syringe	Thermo Fisher Scientific	14-829-9
Dissecting scissors	Thermo Fisher Scientific	08-940
Electrocautery	Thermo Fisher Scientific	50822501
Enclosed incubation chamber	OKOLAB		Microscope
Eye Needles, Size #3; 1/2 Circle, Taper Point, 12 mm Chord Length	Roboz	RS-7983-3
Hank's Balanced Salt Solution	Sigma-Aldrich	55037C
Hoechst 33342	Invitrogen	H3570
Inverted DMi8	Leica		Microscope
Ketamine	Putney		Anesthesia
Kimwipes	VWR	21905-026
McPherson-Vannas scissors 3" (7.5 cm) Long 5X0.15mm Straight Sharp	Roboz	RS-5600
Non-absorbable surgical suture, Silk Spool, Black Braided	Fisher Scientific	NC0798934
Nugent Forceps 4.25" (11 cm) Long Angled Smooth 1.2mm Tip	Roboz	RS-5228
Puralube Vet Ointment	Dechra		Lubricating Eye Ointment
Spinning disk Yokogawa CSU-W1 with a 63x 1.3 N.A. HC PLAN APO glycerol immersion objective, iXon Life 888 EMCCD camera, 405 nm diode laser, 488 nm DPSS laser, 640 nm diode laser	Andor		Confocal system
Xylazine	Akorn		Anesthesia