Assay for the Identification of Interferon-Gamma Secretion in Murine Lymphoid Organs

Overview

This video demonstrates an imaging method to visualize in situ interferon-gamma (IFN-γ) secretion in the mouse spleen. Following bacterial infection and the induction of IFN-γ accumulation inside T cells and natural killer cells, the spleen is harvested from the mouse. Upon obtaining tissue sections, immunostaining is performed to visualize IFN-γ inside the cells.

Protocol

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

1. Adoptive Transfer of Antigenic-specific CD8+ T Cells in Mice

1. Isolate ovalbumin (OVA)-specific CD8⁺ T cells (OTI) expressing green fluorescent protein (OTI-GFP) or red fluorescent protein (OTI-RFP) from lymph node suspension of T-cell receptor transgenic mice using a mouse CD8⁺ T cell isolation kit as per manufacture instructions. Prepare the cell suspension by smashing lymph nodes using a syringe plunger, as previously described.
2. Transfer OTI-GFP or OTI-RFP cells (3 x 10⁶ cells) into C57BL/6 wild-type mice recipients by intravenous injection as described by Cahalan, et al. Use mice that are typically 6–12 weeks of age.
   NOTE: This step is optional and only required for tracking antigen-specific CD8⁺ T cells.

2. Listeria monocytogenes Infection

1. Expand L. monocytogenes genetically modified to express OVA (LM-OVA) to an exponential phase of growth in broth heart infusion at 37 °C under gentle agitation until the OD600 reaches 0.08–0.1, as previously described.
2. Inject 100 µL (maximum volume = 200 µL) of 0.1–0.5 LD50 LM-OVA diluted in phosphate-buffered saline (PBS) by intravenous injection using a 29 G insulin syringe, into C57BL/6 wild-type mice recipients bearing OTI-GFP or OTI-RFP cells when indicated.
   NOTE: In our hands, 0.1 x LD50 LM-OVA corresponds to 2 x 10⁴ colony-forming units (CFU). L. monocytogenes genetically modified to express OVA is used to activate the previously transferred OTI CD8⁺ T cells, but other strains of L. monocytogenes can be used.

3. Treatment with Brefeldin A (BFA) to Block Cytokine Secretion

1. Inject 250 µg of BFA in 200 µL of PBS intraperitoneally for 6 h before mouse sacrifice using a 29 G insulin syringe.
   NOTE: Lyophilized BFA is first resuspended in dimethyl sulfoxide (DMSO) to prepare a stock of 25 mg/mL concentration. The BFA is then diluted in PBS at room temperature (RT) to avoid crystallization prior to injection. Inhibition of cytokine secretion induces accumulation of IFNγ in cells. This is crucial for cytokine detection.

4. Harvesting the Spleen

1. Euthanize the mice using rising concentrations of CO₂ followed by cervical dislocation.
   NOTE: Follow the local institution guidelines for the humane euthanasia of mice.
2. Cleanse the abdomen with 70% ethanol, and make an incision with scissors to make a 1–2 cm cut through the skin on the left flank of the mouse, where the spleen is located. Carefully make an incision in the peritoneum to expose the spleen and take it out with tweezers. Harvest the spleen, being careful not to squeeze it with forceps or cut it to avoid disrupting the spleen architecture.

5. Fixation of the Spleen with Paraformaldehyde (PFA)

1. Prepare the fixative solution by mixing 3.75 mL of PBS and 3.75 mL of 0.2 M L-lysine. Add 21 mg of sodium m-periodate and mix well. Then add 2.5 mL of 4% PFA and 20 µL of 12 N NaOH.
   NOTE: Use the fixative solution on the same day and discard the excess. Do not store it. This fixation step is important if the sample contains fluorescent proteins such as GFP. Do not use PFA containing traces of methanol, as it denatures fluorescent proteins.
   CAUTION: PFA is toxic and must be handled with caution.
2. Submerge the spleen in the fixative and fix for a minimum of 4 h, typically 16–20 h at 4°C under gentle agitation.
3. Discard the fixative solution and add 5 mL of PBS for 5 min at RT under gentle agitation.
4. Replace the PBS with 5 mL of fresh PBS and incubate for 1 h at 4 °C under gentle agitation.
5. Replace the PBS with 5 mL of 30% sucrose, and incubate for 12–24 h.
   NOTE: This method helps maintain tissue morphology. After incubation with sucrose solution, the organ should sink at the bottom of the well.

6. Freezing and Sectioning

1. Put dry ice in a large receptacle and place a smaller receptacle inside containing around 50 mL of pure methanol and a few pieces of dry ice.
2. Gently dry the spleen with a lint-free wipe.
3. Place the spleen inside a base mold containing a drop of optimum cutting temperature (OCT) compound at the bottom. Be careful not to produce any bubbles. Add OCT over the spleen.
4. With forceps, deposit the base mold on the surface of the methanol, making sure that it does not touch the OCT. Freezing the tissue as rapidly as possible to minimize artifacts.
5. When it is frozen, proceed with sectioning.
   NOTE: Frozen spleen can be kept at -80 °C for several months.
6. Section the tissue using a cryo-microtome.
   1. Set the chamber temperature on the cryostat to -21 °C. Cut sections of the desired thickness (usually around 10 µm). This protocol works with thicknesses up to 30 µm.
7. Collect sections onto glass microscope slides (see the Table of Materials) and inspect visually.
   NOTE: Sections can be kept at -80 °C for several months.

7. Immunofluorescent Staining

1. Allow the section to come to RT.
2. Draw a circle with a liquid blocker (e.g., PAP pen) around the tissue section. Draw outside the OCT or it will not stick.
3. Once it has dried, rehydrate the sample by placing PBS on the tissue section for 5 min.
   NOTE: The volume put on the section depends on the size of the section. We typically use 100–300 µL. Do not let the sections dry once they are rehydrated.
4. Rinse with PBS at least twice to ensure that the section is well adhered to the slide.
5. Add a blocking solution to the section to decrease the non-specific binding of antibodies.
   1. Prepare blocking solution as follow: PBS with 0.1% Triton X100, 2% fetal calf serum (FCS), 2.5 µg/mL Fc receptor blocker (anti-mouse CD16/32). Then add 2–5% normal serum of the species of each secondary antibody of the staining panel.
      NOTE: If the antibodies are directly conjugated/biotinylated, add 5% normal serum of the species of each primary antibody. If a primary antibody and one of the secondary antibodies are from the same species (e.g., primary antibody raised in rabbit and secondary rabbit anti-rat), do not use the normal serum species as it will increase the background signal.
   2. Gently remove the PBS from the section by aspiration and add 100 µL of the blocking solution per sample section. Incubate in a covered wet chamber for a minimum of 1 h at RT.
6. Stain with primary antibodies.
   1. Dilute the primary antibodies at the optimal concentration in the blocking solution. The general starting point antibody concentration is 5 µg/mL, but it should be optimized for each antibody and tissue.
      NOTE: If the antibodies are directly conjugated, centrifuge the antibody mix at 17.135 x g (13.500 rpm) for 15 min at 4 °C before using it. Fluorophores can precipitate. This step will pellet the precipitates and thereby prevent non-specific deposition of the precipitated antibodies on the slide.
   2. Replace the blocking solution with the primary antibody mix for each sample.
   3. Incubate for 4 h at RT or overnight (OVN) at 4 °C in a covered wet chamber.
7. Perform washing.
   1. Prepare Wash Buffer by adding 2% FCS to PBS.
   2. Wash 4 times with wash buffer: one quick (without incubation), one for 10 min, and two for 5 min. Then perform a final wash with PBS for 5 min.
8. Staining with secondary antibodies.
   1. Dilute the secondary antibodies of interest at the optimal concentration in the blocking solution. Centrifuge the mix, as described for the primary antibodies.
   2. Remove the final wash solution. Add the secondary antibody mix on top of the section and incubate for 1–4 h at RT in a covered wet chamber.
9. Wash 4 times with wash buffer: one quick (without incubation), one for 10 min, and two for 5 min. Then perform a final wash with PBS for 5 min.
10. Remove the final wash solution. Allow the PBS to evaporate but do not over-dry the section. Place a drop of the mounting medium on top of the sample and carefully place the cover glass on top of it. The mounting medium must recover the whole section. Let it polymerase OVN at RT protected from light.
    NOTE: Draw a circle around the section on the reverse side of the slide before applying the mounting medium. Once the mounting medium is applied, the tissue might become difficult to see.
11. Store the slides in the dark at 4 °C until ready to image.

8. Imaging and Analysis

1. Perform the imaging of the staining with a confocal microscope.
   NOTE: In this protocol, an inverted spectral laser scanning microscope was used (see Table of Materials), together with the objectives 10x/NA 0.40 or 60x/NA 1.4 (for analysis of cytokine sub-cellular localization). Wavelengths of excitation and emission are displayed for each fluorophore and fluorescent protein in the Table of Materials.
2. Perform analysis and quantification as required using image processing software (e.g., Imaris or Fiji).

Materials

Name	Company	Catalog Number	Comments
Brefeldin A	Cambridge bioscience	CAY11861
Paraformaldehyde	Agar scientific	R1018
L-Lysin dihydrochloride	Sigma lifescience	L5751
Sodium meta-periodate	Thermo Scientific	20504
D(+)-saccharose	VWR Chemicals	27480.294
Precision wipes paper Kimtech science	Kimberly-Clark Professional	75512
O.C.T. compound, mounting medium for cryotomy	VWR Chemicals	361603E
Fc block, purified anti-mouse CD16/32, clone 93	Biolegend	101302	Antibody clone and Concentration used: 2.5 mg/ml
Microscope slides - Superfrost Plus	VWR Chemicals	631-0108
anti-CD169 - AF647	Biolegend	142407	Antibody clone and Concentration used: clone 3D6.112 1.6 mg/ml
			Excitation wavelength: 650
			Emission wavelength: 65
anti-F4/80 - APC	Biolegend	123115	Antibody clone and Concentration used: clone BM8 2.5 mg/ml
			Excitation wavelength: 650
			Emission wavelength: 660
anti-B220 - PB	Biolegend	103230	Antibody clone and Concentration used: clone RA3-6B2 1.6 mg/ml
			Excitation wavelength: 410
			Emission wavelength: 455
anti-IFNg - biotin	Biolegend	505804	Antibody clone and Concentration used: clone XMG1.2 5 mg/ml
anti-IFNg - BV421	Biolegend	505829	Antibody clone and Concentration used: clone XMG1.2 5 mg/ml
			Excitation wavelength: 405
			Emission wavelength: 436
anti-Nkp46/NCRI	R&D Systems	AF2225	Antibody clone and Concentration used: goat 2.5 mg/ml
anti-goat IgG-FITC	Novusbio	NPp 1-74814	Antibody clone and Concentration used: 1 mg/ml
			Excitation wavelength: 490
			Emission wavelength: 525
Streptavidin - PE	Biolegend	405203	Antibody clone and Concentration used: 2.5 mg/ml
			Excitation wavelength: 565
			Emission wavelength: 578
streptavidin - FITC	Biolegend	405201	Antibody clone and Concentration used: 2.5 mg/ml
			Excitation wavelength: 490
			Emission wavelength: 525
Fluoromount G	SouthernBiotech	0100-01
Cover glasses 22x40mm	Menzel-Glazer	12352128
Liquid blocker super PAP PEN mini	Axxora	CAC-DAI-PAP-S-M
Imaris - Microscopy Image Analysis Software	Bitplane
Confocal microscope - Olympus FV1200 Laser scanning microscope	Olympus
Cryostat - CM 1900 UV	Leica
Base mould disposable	Fisher Scientific UK Ltd	11670990
PBS 1X	Life Technologies Ltd	20012068
BHI Broth	VWR Brand	303415ZA
GFP			Excitation wavelength: 484
			Emission wavelength: 507
RFP			Excitation wavelength: 558
			Emission wavelength: 583
Insulin syringe, with needle, 29G	VWR International	BDAM324824
C57BL/6 wild type mice	Charles River