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

Podsumowanie

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

Streszczenie

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.

Wprowadzenie

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 for the vaccine development and cancer immunotherapy.

The advent of powerful microscopes - including new confocal and super resolution microscopes - has allowed an extraordinary advancement in understanding how different immune cell populations behave in their native environment³. It is now possible to image several simultaneous cell subtypes using a combination of probes with genetically modified mice that express fluorescent proteins under control of specific targets^4,5. In fact, high dimensional techniques, including mass cytometry and multi-parametric flow analysis have been crucial to expanding our knowledge on different immune cell compartmentalization and functionality in the health and disease^6,7. However, to prepare samples for these techniques, tissues need digestion and cells are separated from their natural milieu to be analyzed in cell suspensions. To surpass these limitations and allow a better translation in biology, the goal of the protocol proposed here is to apply a straightforward methodology to image ex vivo whole lymph nodes using stock confocal microscopes with the benefit of improved speed, tissue structure preservation, and cell viability compared to the conventional immunofluorescence staining. By using this approach, we were able to show that mice deficient for γδ T cells, a subtype of T lymphocyte involved in host early defense against pathogens⁴, have compromised follicles and T cell zones as compared to wild type mice. These findings allowed us to pursue a study in which we demonstrated that γδ T cells play a critical role in the homeostasis of lymphoid organs and humoral immune response⁴. Furthermore, this protocol provides a physiologic pathway for probes and antibodies to reach the lymph node, as they are administered subcutaneously and dissipate through the tissue lymphatic circulation, building on previous reports that used in situ labeling with antibodies to visualize lymphatic-associated structures^8,9, germinal center dynamics^10,11,12, and targets readily accessible to blood flow^13,14,15.

Protokół

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 the presence of mononuclear cells, including antigen presenting cells and phagocytes, and their distribution in the LN.
   NOTE: CX3CR1^GFP/WTCCR2^RFP/WT reporter mice have green fluorescent protein (GFP) and red fluorescent protein (RFP) inserted under the control of CX3CR1 and CCR2 promoters, respectively. Reporter mice can be used with or without the injection of the antibody mix. Please see reference⁴ for antibody mix injection in a reporter mouse. Proceed to surgery if no antibody will be injected.

2. Antibody mix preparation and injection

NOTE: Perform these steps on mice described in step 1.1.

1. Dilute 1:10 of brilliant violet (BV) 421 anti-CD4 (GK1.5; 0.2 mg/mL), 1:10 of brilliant blue (BB) 515 anti-CD19 (1D3; 0.2 mg/mL) and 1:20 of phycoerythrin (PE) anti-F4/80 (T45-2342; 0.2 mg/mL) in PBS with the appropriate final volume to inject into the inner thigh (to image inguinal) or into the paw pad (to image popliteal) lymph.
   NOTE: Use as isotypes: BV421 Mouse IgG2b, k Isotype Control (R35-38; 0.2 mg/mL); PE Rat IgG2a, κ Isotype Control (R35-95; 0.2 mg/mL); BB515 Rat IgG2a, κ Isotype Control (R35-95; 0.2 mg/mL). If changing the staining antibody, use correct isotype.
2. To image inguinal LN, inject 100 µL of the antibody mix subcutaneously into the inner thigh (Figure 1A). Alternatively, inject 50 µL subcutaneously of antibody mix into the paw pad to image popliteal LN (Figure 2A). Use delicate 1 mL Insulin syringes, (Insulin U-100) with the needle of size 0.30 mm × 13 mm (30 gauge × ½ inch).
   NOTE: Ensure that the injection for inguinal LN staining is subcutaneous and not intramuscular (i.m.), as antibody mix will not be properly drained if i.m. administration is performed.
3. Do not anesthetize animals before antibody mix injection.
4. Wait for a minimum of 3 h (inguinal dLN) and 12 h (popliteal dLN) post injection to remove the organs.
5. If LN cell labeling is not completely observed using large polymer fluorescent dyes such as brilliant violet or brilliant blue, use smaller fluorophores, including fluorescein isothiocyanate (FITC), PE and allophycocyanin (APC) as an alternative.

3. Surgery procedure to remove the inguinal draining lymph node

1. Euthanize mice using CO₂ asphyxiation followed by cervical dislocation.
2. Immobilize mice on the acrylic stage with adhesive tape and apply mineral oil with a cotton swab to the abdominal skin to prevent fur deposition around the incision (Figure 1B). Fur removal is not necessary.
3. Perform a midline incision using microsurgery curved scissors (11.5 cm) and microsurgery curved forceps (12.5 cm) in the abdomen from the pubis to the xiphoid process (Figure 1C).
4. Dissociate the abdominal musculature from the skin (Figure 1D).
5. Make horizontal skin incisions at the top and bottom of the vertical incision line to create skin flaps on side of interest (according to the side of the antibody mix injection) and flap the skin to visualize the lymph node (Figure 1E).
6. Tape the skin-flap on the acrylic plate (Figure 1F).
7. Remove the inguinal draining lymph node using microsurgery curved forceps (Figure 1F). Lymph node will appear as a translucid, usually bilobular, sphere under the skin.

4. Surgery procedure to remove the popliteal draining lymph node

1. Euthanize mice using CO₂ asphyxiation followed by cervical dislocation.
2. Immobilize mice at a prone position on acrylic stage with adhesive tape and apply mineral oil with a cotton swab in the calf and knee (Figure 2A-D).
3. Perform a midline incision in the calf from the heel to the knee (Figure 2E,F).
4. Dissociate the calf musculature from the skin (Figure 2F,G).
5. Expose the popliteal fossa (Figure 2G). Popliteal lymph node will appear as a translucid sphere in the popliteal fossa.
6. Remove the popliteal lymph node using microsurgery curved forceps (Figure 2G).
7. Alternatively, turn the mouse over on supine position, and approach the popliteal fossa between biceps femoris and semitendinosus for popliteal LN removal by performing a midline incision in the calf from the heel to the knee followed by dissociation of the calf musculature from the skin.
   NOTE: Popliteal fossa is a shallow depression located at the back of the knee joint. Open carefully to see the popliteal lymph node.

5. Lymph node preparation

1. Place the whole organ in a culture dish with glass bottom (35 mm x 10 mm) and remove the fat that surrounds the organ using microsurgery curved forceps (Figure 1G-I and Figure 2H).
2. Centralize the organ in the middle of the dish (Figure 1J and Figure 2H).
3. Cover the organ with a fragment of delicate task wipers and keep it soaked with room temperature saline 0.9% or Phosphate Buffer Solution (Figure 1K-M and Figure 2H).
   NOTE: It is not necessary to wash lymph nodes after removal or to perform the lymph node extraction in the hood.

6. Ex-vivo confocal microscopy (Imaging)

1. Position the Petri dish in the inverted confocal microscope slot (Figure 1N and Figure 2H).
2. Image LNs under a confocal microscope (Figure 1O and Figure 2H).
3. First, obtain the correct focus by using the conventional light of the confocal microscope with 4x or 10x objective. Then change from the light function to the laser mode.
   NOTE: If the confocal microscope does not contain a 4x objective, the focus can be perfectly obtained using the 10x objective.
4. Adjust the laser power, offset and gain using an isotype-stained, non-stained or non-fluorescent sample (Table 1) to remove autofluorescence and unspecific staining from fluorescent-labeled antibodies such as the ones used in the images showed in this manuscript (BV-421 anti-CD4, BB515 anti-CD19 and PE anti-F4/80).
5. Adjust Z and XY positions, using the micrometric and chariot, respectively, on the area of interest in the lymph node.
6. Acquire images under 4x, 10x and 20x objectives focusing on the LN structure and cellular distribution. Use 1024 x 1024-pixel definition.
   NOTE: Acquire a minimum of five images in different fields per objective per animal.
7. Analyze images using an image software to separate channels, add scale and colors of interest, and to reconstruct 3D view.

Wyniki

This manuscript shows techniques to remove inguinal and popliteal lymph nodes without damaging their structure following the injection of fluorescent-labeled antibodies to stain specific cell populations in these organs (Figure 1 and Figure 2).

The powerful combination of immunolabeling of LN cells with BV421 anti-CD4 and BB515 anti-CD19 and confocal imaging analysis defined the localization of T cells (CD4+) and B cells (CD19+) in in...

Dyskusje

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...

Materiały

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)