The overall goal of this procedure is to determine the location, abundance and phenotype of antigen-specific CD8 positive T cells within tissues of interest. This method helps answer quick questions about CD8 T cells by providing a means to locate, quantify and phenotype virtually any antigen specific CD8 T cell and any tissue. The main advantage of this technique is that it allows determination of the spatial distribution of antigen specific CD8 positive T cells relative to other cells and structures within tissues of interest.
Demonstrating the procedure with Shengbin Li will be Gwantwa Mwakalundwa. On the day one of the procedure, use a scalpel to cut the fresh tissue into approximately 0.5 x 0.5 centimeter pieces. Here, rhesus macaque spleen tissue is shown.
Separately glue each tissue fragment into the middle of the top of a plunger and embed the samples in three to five milliliters of warm 4%low melting agarose in PBS. Label the plungers with the relevant tissue information. Then, place the samples in a chilled holder in an ice bucket.
When the agar has solidified, turn on the microtome and set the thickness setting to 200 micrometers. After installing a new razor blade, insert the plungers into the microtome bath. Fill the microtome bath with 100 to 200 milliliters of freshly prepared PBS-H and add PBS-H ice cubes to maintain the temperature between zero to two degrees celsius.
After obtaining 200 micrometer sections, use a paintbrush to remove the agar surrounding each section and transfer the samples into individual tissue chambers within individual wells of a 24 well tissue culture plate containing one milliliter of chilled PBS-H. When all of the samples have been acquired, incubate the tissue sections overnight in one milliliter of fit conjugated peptide loaded MHC class one tetramers, diluted in PBS-H supplemented with 2%normal goat serum at four degree celsius with rocking. The following morning, transfer each tissue chamber into individual wells of a new 24 well plate containing one milliliter of fresh chilled PBS-H for 20 minutes.
After the second wash, fix the sections in one milliliter of fresh 4%paraformaldehyde for two hours at room temperature. Followed by two five minute washes in cold PBS-H. Transfer the sections into a new 24 well plate containing 0.01 molar urea and microwave the sections three times for about 10 seconds each time until the urea boils.
To permeabilize and block the samples, incubate the sections in blocking solution containing PBS-H supplemented with a 0.3%triton x100 detergent and 2%normal goat serum on a rocker for one hour, at four degree celsius. At the end of the incubation, transfer the tissue chambers into individual wells of a new 24 well plate containing one milliliter of solution containing rabbit anti-fitc antibody, mouse antihuman CD antibody and rat antihuman CD3 antibody. Incubate the samples overnight at four degrees celsius with rocking.
On day three, wash the sections three times in fresh PBS-H at four degree celsius for at least 20 minutes per wash and perform a final incubation with the appropriate fluorescence conjugated secondary antibodies for up to three days, protected from light. At the end of the tertiary antibody incubation, rinse the samples with three 20 minute washes in fresh PBS-H and use a paint brush to transfer the samples from each tissue chamber onto a glass microscope slide. Coat each section with mounting medium supplemented with a fluorophore preservative and cover the samples with a cover slip.
Then, store the slides in a light protected slide container, at 20 degrees celsius. On the day of imaging, place the first slide onto the stage of a confocal microscope and select the appropriate lasers and filters for each fluorophore used to label the tissue sample. Using the 20x objective, sequentially obtain a z series at three micron intervals in each of the fluorophore channels, in multiple 800 x 800 pixel fields.
Then, construct a montage of the collected fields and save each montage image with a name based on the corresponding tissue sample. In these representative images, a section stained with MHC class one tetramers loaded with an SIV peptide is compared to a section from the same tissue, stained with MHC class one tetramers loaded with an irrelevant peptide, demonstrating the specificity of the tetramer staining. In these images, the virus specific T cells stained with MHC class one tetramer loaded with SIV peptide, co-stains for CD8, a T cell subset marker but not CD20, a cell surface protein expressed on B cells.
This montage was created for multiple confocal z series fields, that were used for the quantification of tetramer stained cells with specific phenotypes and different anatomical compartments on the lymph node. This magnified image highlights an area of the montage lymph node that contains a B cell follicle delineated by IGM staining. MHC class one tetramer stained cells, some of which co-expressed the T cell activation and proliferation marker, KI67 can be detected inside and outside of B cell follicle.
This staining combination allows the determination of the activation and proliferation statuses of SIV specific CD8 positive T cells, as well as their localisation within secondary lymphoid tissues. Once mastered, this technique can be completed in approximately five days if it is performed properly. While attempting this procedure, it's important to remember that fresh tissue is critical for successful staining.
Process the samples within 24 hours or less after harvesting, keeping them chilled until fixation. After watching this video, you should have a good understanding of how to perform a tetramer staining and immunohistochemistry to determine the location, abundance and phenotype of antigen specific CD8 positive T cells in tissues of interest. Don't forget that working with infectious agents can be extremely hazardous and that precautions to prevent exposure such as, wearing the appropriate personal protection equipment and limiting the use of sharps should always be taken while performing this procedure.
