Intravital Visualization of Gamma Delta Intraepithelial Lymphocytes
Published: August 31, 2023
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. Perform experiments on 8–10 weeks …
Representative 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.
Declarações
The authors have nothing to disclose.
Materials
| 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 |
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Citar este artigo
Intravital Visualization of Gamma Delta Intraepithelial Lymphocytes. J. Vis. Exp. (Pending Publication), e21569, doi: (2023).