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

Interferon-gamma, or IFN-γ, a cytokine secreted by the lymphocytes T cells and natural killer cells or NK cells, mediates the immune response against pathogens.

To study in situ IFN-γ production, take a mouse transplanted with fluorescently-labeled T cells engineered to express a receptor recognizing ovalbumin — a model antigen.

Take a suspension of pathogenic bacteria — genetically modified to express ovalbumin — and inject it intravenously into the mouse, causing a systemic infection. The circulating bacteria reach the spleen — a secondary lymphoid organ — which aids pathogen removal by facilitating interaction with immune cells.

Antigen-presenting cells engulf the bacteria and display the antigen. T cells bind to the antigen via its receptor, are activated, and produce IFN-γ.

Macrophage cells, infected with the bacteria, release immune mediators and upregulate activating ligands on their surface. Upon interaction with these signals, NK cells get activated and produce IFN-γ.

Inject an intracellular protein transport inhibitor to restrict IFN-γ secretion, causing its intracellular accumulation.

Harvest the mouse spleen. Fix the tissue and freeze it to aid in sectioning. Using a cryo-microtome, generate thin tissue sections, and place them on glass slides. Add NK cell-specific antibodies, which bind to the surface markers, and IFN-γ-specific antibodies, which stain the intracellular IFN-γ. These antibodies are labeled with different fluorophores.

Place the slide under a fluorescence microscope to detect IFN-γ inside the NK cells and T cells.

Discard the fixative solution, and add 5 milliliters of PBS for 5 minutes at room temperature, under gentle agitation. Then, replace the PBS with 5 milliliters of 30% sucrose and incubate for 12 to 24 hours to maintain the tissue morphology. Now, the organ sinks at the bottom of the plate.

To freeze the sample, put dry ice in a large receptacle and place a smaller receptacle inside, containing around 50 milliliters of pure methanol and a few pieces of dry ice. Gently dry the spleen on a lint-free wipe. Drip a drop of OCT compound at the bottom of the base mold and place the spleen inside the mold. Be careful not to produce any bubbles.

Add approximately 1 milliliter of OCT over the spleen. With forceps, deposit the base mold on the surface of the methanol in the dry ice bath, making sure the methanol does not touch the OCT. The OCT will thicken and become white when frozen. Remember to freeze the tissue as rapidly as possible to minimize artifacts.

On a cryo-microtome, set to the temperature of minus 21 degrees Celsius. Section the tissue to desired thickness around 10 micrometers. Use a brush to collect sections onto glass microscope slides, and inspect visually. Allow the sections to come to room temperature.

Draw a circle with a liquid blocker, for example, a PAP pen, around the tissue section outside the OCT. Once the tissue has dried, rehydrate the sample by dripping 100 microliters of PBS on the tissue section for 5 minutes. Then, remove the PBS from the section by aspiration, and add 100 microliters of blocking solution per sample section. Incubate in a covered wet chamber for a minimum of one hour at room temperature.

To stain with primary antibodies, replace the blocking solution with the prepared primary antibody mix for each sample. Incubate for four hours at room temperature or overnight at 4 degrees Celsius in a covered wet chamber, before washing according to the manuscript.

Now, dilute the secondary antibodies of interest to an optimum concentration, usually 2.5 micrograms per milliliter in the blocking solution. Remove the final wash solution from the section, and add the prepared secondary antibody mix on top of the section, and incubate for one to four hours at room temperature in a covered, wet chamber.

After washing with wash buffer according to the manuscript, remove the wash solution and perform a final wash with PBS. Aspirate the phosphate-buffered saline and allow the remaining PBS to evaporate without the section completely drying out.

Draw a circle around the section on the back of the slide. Then, place a drop of the mounting medium on top of the sample, making sure the medium covers the whole section, and carefully place a cover glass on top. Let the samples polymerize overnight at room temperature in the dark.

In the morning, place the slide under an inverted spectral laser-scanning microscope. Adjust the objectives to 10x/NA 0.40, or 60x/NA 1.4 for analysis of cytokines sub-cellular localization.