This protocol is significant because it allows us to visualize the microenvironment where cells produce a given cytokine, here, interferon gamma. It is admitted that the range of action of cytokines is limited because tissues are so complex. It is important to characterize the cells that surround interferon gamma-producing cells to pinpoint which cells will be taking it up.
The main advantage of the technique is that it does not disrupt the microenvironment present in the tissue of interest. In addition, because we do not artificially restimulate cells to force them to produce interferon gamma, it gives us a better characterization of whether cells are actually actively producing interferon gamma in situ. This protocol could be potentially adapted to other organs and for the visualization of other cytokines.
Most of the steps in this protocol are simple, but handling of the tissue, freezing and sectioning, should be performed with care to preserve the integrity of the tissue. To begin, transfer 300 microliters of LM cultured genetically modified to expressed OVA to a broth heart infusion at 37 degree Celsius in a flask. Place the flask on a shaker for gentle agitation to grow LM to an exponential phase until the OD600 reaches 0.08 to 0.1.
After diluting the LM OVA culture in PBS to a concentration of 0.1 times lethal dose, 50%use a 29-gauge insulin syringe to intravenously inject 100 microliters into C57 black 6 wild type mice recipients bearing OT1 GFP or OT1 RFP cells. To block cytokine secretion 6 hours before mouse sacrifice, inject 250 micrograms of BFA and 200 microliters of PBS intraperitoneally with the 29-gauge insulin syringe. After euthanization of mice using carbon dioxide and subsequent cervical dislocation, cleanse the abdomen with 70%ethanol.
On the left flank of the mouse where the spleen is located, cut through the skin with scissors to make an incision of one to two centimeters. Then, carefully make an incision in the peritoneum to expose the spleen and take it out with tweezers. Be careful not to squeeze it with forceps or cut it to avoid disrupting the spleen architecture.
Now, prepare the fixative solution by mixing 3.75 milliliters of PBS and 3.75 milliliters of 0.2 molar L-lysine. Add 21 milligrams of sodium m-periodate and mix well. Then add 2.5 milliliters of 4%PFA and 20 microliters of 12 normal sodium hydroxide.
Cut the spleen into three parts. Submerge the spleen in the fixative in a six-well plate and fix for a minimum of four hours. A typical fixation period is 16 to 20 hours at four degree Celsius under gentle agitation.
After that, discard the fixative solution and add five milliliters of PBS for five minutes at room temperature under gentle agitation. Then, replace the PBS with five 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 one 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 20 degree 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 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 five 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 four degree 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 sample polymerase overnight at room temperature in the dark. In the morning, place the slide under an inverted spectral laser scanning microscope. Adjust the objective to 10x NA 0.40 or 60x NA 1.4 for analysis of cytokine subcellular localization.
In this protocol, cells that produce interferon gamma are visualized and located. The marker F4/80 labels all macrophages and highlights the red pulp. The marker B220 labels B cells and highlights B cell follicles surrounding the T-cell zone.
The marker CD169 labels marginal zone macrophages surrounding the white pulp. 24 hours after infection, interferon gamma is produced by multiple cells, including activated antigen-specific OT1 CD8 T-cells and NK cells. Without the use of BFA to inhibit cytokine secretion, the detection of interferon gamma by NK cells was greatly impaired.
B cells, marginal zone macrophages, and all macrophages were stained to delineate the location of interferon gamma producing cells following LM OVA infection. Interestingly, clustered antigen specific T-cells were found located throughout the white pulp of the spleen, but they produce interferon gamma only in regions where NK cells were coexisting with them. Different subcellular localization of interferon gamma and NK versus CD8 positive T-cells was shown.
While interferon gamma localization in NK cells was diffused in the cytosol, CD8 positive T-cells often recruited interferon gamma towards another T-cell. The injection of brefeldin A is crucial to detect the cytokine in situ. Cells often quickly secrete and take up the cytokines and we need to inhibit cytokine secretion to let it accumulate in cells.
This method is a discovery tool that places back the cytokine producing cells in their native environment. Deciphering the composition of this environment allows us to focus on the right cells or immune mediators present at this location that can impact or be impacted by interferon gamma. There is a growing understanding that the precise localization of a cell in a tissue is crucial for its function.
Our protocol allows us to identify the localization of the cytokine producing cells and thereby, further characterize its function.
