By combining in situ hybridization and tetra staining in a technique known as ISTH, the relative numbers and spatial relationships between viral antigen specific immune effector cells and virally infected cells and tissues can be determined. ISTH begins by embedding fresh SIV infected tissue in agros. The tissue is cut into 200 micron thick sections using a vibrato and stained for viral antigen.
Specific CD eight positive cells using fit C labeled MHC tetramers. After freezing, these stain sections are further cut into eight micrometer thick sections using a cryostat and in situ hybridization is performed to localize virally infected cells. Confocal microscopy is used to image the double stain sections and ultimately reconstruct the original thick sections to reveal the relationship between immune effector and target cells.
Using this procedure for examining early SIV infection has proved to be a good predictor for the severity of the ensuing disease. Hi, I'm Chinen Lee from the lab tree of Ashley HA in the Department of Microbiology at the University of Minnesota. I'm Pam Skinner, an assistant professor in the Department of Veterinarian Biomedical Sciences at the University of Minnesota.
Today we will show you a procedure for inside in the institute hybridization or ISTH for short. In addition to using inci teer staining combined with inci hybridization as we're demonstrating today, we also routinely use inci teer staining combined with immunohistochemistry to determine the location, abundance and protein expression profile of antigen specific CD eight T cells in situ you. Assisting us today with the in situ you temer staining with the thick sections will be Dr.Heon Kim and Terry Matala and assisting us with a thin section preparation and in situ you hybridization will be So let's get started.
To begin this procedure, chill the Vibram bath containing sterile PBSH to naugh to two degrees Celsius. Set the vibrator blade angle to 27 degrees. Whenever possible, keep fresh tissues chilled on ice and begin procedure within 24 hours after harvesting tissues.
In order to minimize degradation chilling, the vibram also facilitates cutting a fresh tissue, trim away connective tissue and cut the specimen with scalpel into small pieces and put the pieces in a whey boat and cover with 4%low melt. Agros in PBS melted at around 40 degrees Celsius. Make sure the tissue is in contact with the bottom of the dish and push tissue pieces down if they float, solidify on ice for three to five minutes.
Here spleen tissue is being used, but lymph node, vagina, cervix, uterus or other fresh tissues can also be used with a scalpel. Cut the agros around the tissue and using forceps carefully transfer the agros embedded tissue to the tissue block. Continue by coating a Vibram tissue block with Loctite glue.
Do not move the piece of tissue once it is set on the tissue block. Let dry for about 10 minutes on ice. Next, mount the tissue block in the vibrator and cut the tissue into 200 micrometer thick sections using a dead slow forward speed and relatively high amplitude.
Use a paintbrush to transfer the cut sections into a specialized tissue chamber set in the well of a 24 well tissue culture plate prefilled with one milliliter of chilled PBSH. Put up to four tissue sections into each tissue chamber. Label the samples and secure the lid to the plate with a rubber band in order to stain the tissue sections for viral specific T cells.
ZI conjugated tetramers are first diluted in blocking buffer to nwt 0.5 micrograms per milliliter. The solution is pipetted into a 24 well plate at one milliliter per well. The freshly cut tissue is transferred to the plate in the tissue chambers and is allowed to rock overnight at four degrees Celsius.
Keep an empty well between tissue chambers containing different samples to prevent cross contamination of solutions in subsequent steps. At this step one can add antibodies directed against extracellular or cell surface proteins. For washing, prepare a 24 well tissue culture plate containing chilled PBSH after the primary incubation.
Washed twice by transferring the tissue chambers to a prefilled plate for 20 minutes each time, be sure to drain most of the liquid from the tissue chambers before transferring. Also, do not drip content of one sample into another when moving the tissue chambers and monitor the sections in the tissue chambers to make sure that they do not get stuck to the sides. Fix the sections with fresh 4%paraldehyde in PBS for two hours at room temperature after the fixation.
Washed twice with cold PBSH for five minutes each time to amplify the signal from the tetramers. Incubate the sections with rabbit anti fitzy antibodies at a ratio of one to 10, 000 in blocking buffer for one to three days. At this stage, one can add antibodies against intracellular epitopes.
Making sure to perme alize the cells and block nonspecific sites prior to the addition of the antibody. Antigen retrieval may be required as well after the second long incubation intended to amplify the signal. Wash the sections in PBSH three times for at least 20 minutes and up to several hours each time.
Perform a final incubation with appropriate fluorescently labeled antibodies. For example, one may use goat anti rabbit SI three to bind the rabbit anti fitzy antibodies to make the tetramer stain cells fluoresce red. If mouse antibodies were used to counterstain proteins of interest such as CD eight to visualize killer T cells one may use goat anti-US Alexa 488 to bind into the mouse antibodies and make the antibody stain cells fluoresce green dilutions of each antibody must be determined empirically.
Incubate for one to three days keep sections protected from light by wrapping the plates and tinfoil during this incubation and thereafter as light quenches flora falls. Finally fix the sections again to secure the tetramers and the antibodies in place. Wash and mount on a microscope slide with glycerol gelatin containing four milligrams per milliliter N profile gallate for storage at minus 20 degrees Celsius until preparing thin sections for in-situ hybridization.
The insitu tetramer stain sections can now be analyzed with a confocal microscope in order to determine the localization abundance and protein expression pattern of antigen specific CD eight positive T cells in the image shown spleen tissue sections from an SIV infected recess meac was stained with gag tetramers to label SIV specific CD eight positive T cells CD 20 antibodies and CD three antibodies. Since counters staining with antibodies will be lost in the next step. It is important to analyze in situ tetramers before proceeding forward.
Begin the thin sectioning procedure by heating the 200 micron thick sections at 70 degrees Celsius for five minutes on a heat block in order to detach thick sections from the slide. Once the sections are detached, embed them in OCT on dry ice by first filling the bottom of the plastic mold with OCT. Then place the tissue on top of the OCT and cover it with OCT.
Use a cryostat to cut eight micron thick sections from the OCT embedded sections and adhere the eight micron sections to siloized slides. The slides can then be stored at minus 80 degrees Celsius in a sealed box for subsequent in situ hybridization. Standard in situ hybridization is used to detect virally infected cells via S 35 labeled virus specific rib probes.
The procedure also includes coating sections with nuclear track emulsion and mounting slides. In a fluorescent compatible paramount medium confocal images are acquired using a BioRad MRC 1000 confocal microscope, which is equipped with an epi tube blue reflection device. The insitu tetra staining is visualized using the EPI one 605 for the red tetra fluorochrome and EPI 2 522 DF 32 for the green CD eight fluorochrome for insitu hybridization.
Use the EPI two blue reflection for the silver grains resulting from the in-situ hybridization signal in the developed radio autographs. To begin image acquisition, acquire the images from each section from left to right and top to bottom and make sure to capture each image with an approximately 20%overlap with its neighboring images. To avoid gaps in the reconstructed montage image, name each image according to its bro and column in an Excel file.
When data collection from the slides is complete, use the confocal assistant software to project Z serial images for each channel into a single image using a Photoshop seven action procedure available from the authors, automatically merge the projected image from the three channels into one RGB image. Then stitch the merged images together in layers to generate a montage image of the whole section. Associate individual silver grains and tetramer stain with cells in the montage using metamorph or Photoshop.
Assign the XY coordinates of the centers or OIDs of the viral RNA positive and tetramer positive cells. Export the OID data into Excel files as numeric numbers for each cell and use the data collected in the Excel files to determine the ratios of immune effector or e cells to viral targets or T cells from the total numbers of tetramer positive and viral RNA positive cells in section to capture the spatial relationships between tetramer positive and RNA positive cells. Copy and paste their respective plots from the Excel files into a Photoshop document in Photoshop.
Decrease the opacity of a layer to align and scale the grids so that they coincide color, select, and then copy and paste into a new layer. The positions of the blue RNA positive cells discard the layer used to align the grids and the positions of the tetramer positive and viral RNA positive cells will be revealed in the flattened image. Using the ISTH method shown here, we are able to follow the immune response to SIV infection following intravaginal inoculation at this portal of entry and in the lymphatic tissues to which infection spreads.
There are large numbers of infected cells at these tissue sites at 10 days post-infection, but as late as three days later, there are green stain CD A positive cells, but not as yet. Red stained SIV specific tetra positive cells. The immune response is thus too late to clear the infection.
However, at three weeks after infection, a decrease in infected cells is clearly visible, especially at the portal of entry. We used ISTH to show that the extent of the reduction was correlated with spatial proximity and relative numbers of tetramer positive immune effector cells. N-S-I-V-R-N-A positive target cells, we call this the in vivo E to T ratio.
The spatial relationships are readily visualized using this method with effector cells in red and viral targets in blue in this infected lymph node. Many effector and target cells are in close proximity and there are areas where many effector cells surround a single S-I-V-R-N-A positive cell while in other areas an S-I-V-R-N-A positive cell stands alone. The ratio of ETT is simply calculated as the positions of the cells are determined and logged in by the numbers of each in the Excel file.
The best control of the viral infection correlates with a great excess in red effectors over green targets 43 to one. In this case, in the cervix and vagina where infection started ETT ratios were lower in lymph nodes. The positive correlation between high ETT ratios and reduction of viral load from peak indicates that a more robust immune response at the portal of entry translates into better control of infection.
We have just show you how to simultaneously detect the very specific CDAT cells and the virus infection cell in situ. This technique allows us to simultaneously visualize antigen specific T cells and virus infected cells in tissues. This technique can be applied to evaluate the CD eight positive T-cell based vaccine and the other non-value pathogen and host interaction When doing this procedure.
It's important to keep all reagents RNAs free prior to the insights you hybridization. So that's it. Thanks for watching and good luck with your experiments.
