Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy

Summary

This protocol describes a technique to image different cell populations in draining lymph nodes without alterations in the organ structure.

Abstract

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are strategically interposed in the path of the lymphatic vessels, allowing intimate contact of tissue antigens with all resident immune cells in the LN. Thus, understanding the cellular composition, distribution, location and interaction using ex vivo whole LN imaging will add to the knowledge on how the body coordinates local and systemic immune responses. This protocol shows an ex vivo imaging strategy following an in vivo administration of fluorescent-labeled antibodies that allows a very reproducible and easy-to-perform methodology by using conventional confocal microscopes and stock reagents. Through subcutaneous injection of antibodies, it is possible to label different cell populations in draining LNs without affecting tissue structures that can be potentially damaged by a conventional immunofluorescence microscopy technique.

Introduction

Lymph nodes (LNs) are ovoid-shaped organs widely present throughout the body with the crucial function of bridging the innate and adaptive immune responses. LNs filter the lymph in order to identify foreign particles and cancerous cells to mount an immune response against them¹. Antigen presenting cells (APCs), T cells and B cells work alongside to generate antigen-specific antibodies (humoral immunity) and cytotoxic lymphocytes (cellular immunity) to eliminate the foreign particles and cancerous cells². Thus, understanding the dynamics of the immune cells present in the lymphatic system will have important implications ....

Protocol

The protocol was approved by the Standing Committee on Animals at Harvard Medical School and Brigham and Women’s Hospital, protocol 2016N000230.

1. Mice used for the experiment

1. Use 8-week old male and female mice on the B6 background for administering the antibody mix.
2. Use CX3CR1^GFP/WTCCR2^RFP/WT mice to determine whether ex vivo whole LN imaging can also be applied to reporter mice without administering antibody mix as well as to investigate th.......

Discussion

The combination of imaging with other techniques, including molecular biology and high dimensional immunophenotyping has enhanced our ability to investigate immune cells in their native context. In fact, while other approaches may require tissue digestion and cell isolation – which can lead to loss of tissue integrity - the use of in vivo or ex vivo imaging grants a great advantage in investigating different cell subtypes in a geographical fashion^3,16. It i.......

Materials

Name	Company	Catalog Number	Comments
BV421 anti-CD4	BD Horizon	562891	GK1.5; 0.2 mg mL-1
BB515 anti-CD19	BD Horizon	564509	1D3; 0.2 mg mL-1
BB515 Rat IgG2a, κ Isotype Control	BD Horizon	564418	R35-95; 0.2 mg mL-1
BV421 Mouse IgG2b, K Isotype Control	BD Horizon	562603	R35-38 0.2 mg mL-1
Cellview culture dish	Greiner-Bio	627861	35x10 mm with glass bottom
Insulin syringes	BD Plastipak	-	Insulin U-100
Kimwipes	Kimtech Science Brand	7557	size 21 x 20 cm / 100 sheets per box
Microsurgery curved forceps	WEP Surgical Instruments	custom made	12.5 cm
Microsurgery curved scissors	WEP Surgical Instruments	custom made	11.5 cm
Needle	BD PrecisionGlide	-	30 gauge × ½ inch
Nikon Eclipse Te + A1R confocal head	Nikon	-	loaded with main 4 laser lines (405, 488, 543 and 647 nm)
PE anti-F4/80	BD Pharmigen	565410	T45-2342; 0.2 mg mL-1
PE Rat IgG2a, κ Isotype Control	BD Pharmigen	553930	R35-95; 0.2 mg mL-1
Zeiss LSM 710 confocal microscope	Zeiss	-	loaded with main 4 laser lines (405, 488, 543 and 647 nm)