The overall goal of this procedure is to detect self reactive T cells with a high degree of specificity using MHC Class two dex trimmers. In this video, cerebral is harvested from the brain of a myelin protio lipid protein induced experimental autoimmune encephalomyelitis mouse. This is accomplished by first carefully harvesting the tissues and preparing the aros blocks.
The second step is to cut the aros embedded tissue into 200 micrometer thick sections with a viome. The sections are then transferred to tissue chambers in a 24 well plate where they are stained and fixed. The final step is to mount the tissue sections on microscopic slides.
Ultimately, confocal microscopy is used to show the cells bound with dex trimmers and CD four antibody. We first conceived this idea when we first demonstrated that the MHC class two dextro ERs were more sensitive than the conventional MHC Class two tetramers for detecting the agen specific self reactive CD four T cells. Generally individuals new to this method will struggle because sectioning of fresh tissues and their handling is fairly challenging.
Visual demonstration of this method is critical as the sectioning mounting and analysis steps are difficult to learn because they are tricky. In addition to Ting two postdoctoral research associates, Chandy and Aaron from my group and Christian from Dr.Zoe's laboratory will be demonstrating this procedure Following the euthanasia of experimental autoimmune encephalomyelitis or EAE mice excavate the skull by snipping the cranium in a circular fashion using a pair of curved scissors and carefully flip the skull bones with forceps. Harvest the brain by blunt dissection using a clean scalpel.
Separate the rerum from the cerebellum. Place the rerum in a tube containing cold one x phosphate buffered saline or PBS and leave the tube on ice until processing. Place the rerum in a disposable deep base mold histology cassette kept over ice.
Then pour molten 4%PBS buffered low melting aros into the cassette. Immediately press the cerebrum gently with forceps to submerge the tissue and leave the cassette on ice until the solidification is complete. Once solidified, trim excess aeros from all the sides of the embedded tissue and slightly expose the ventral surface of the rerum.
Next, smear the Vibram tissue block with super glue and transfer the aros embedded tissue over the glued surface by placing the exposed ventral surface of the cerebro over the glue for transverse sectioning. Do not move the tissue once it is set over the block. Place the vibrate tone block containing the aros embedded tissue on ice and allow the glue to dry for 15 minutes.
In the meantime, place prepared tissue chambers into a 24 well plate at one chamber per well. Fill the wells containing the tissue chambers with one milliliter of cold one XPBS and leave the plate on ice. Once the glue is dry, lower the vibram blade to the level of the top surface of tissue.
Set the Vibram speed to 0.22 millimeters per second and start sectioning with a forward movement to make 200 micrometer thick sections. Using a watercolor soft brush transfer one cerebral section into each tissue chamber. In the 24 well plate, keep the plate on ice until sectioning is complete to prevent tissue degradation.
Next, prepare a second 24 well plate by adding 300 microliters of a cocktail containing IA of S myelin protio lipid protein 1 39 to 1 51 dextro and CD four antibody to three of the wells. Then add 300 microliters of the control IA of S Tyler's murine encephalomyelitis virus 70 to 86 dextro and CD four antibody cocktail to another set of three wells. Proceed to label the wells.
Then transfer three tissue chambers containing the sections designated for staining with specific dex trimmers into the three designated wells of the 24 well plate with one chamber per well. Similarly, transfer the three tissue chambers containing the sections designated for staining with controlled dex streamers into those wells. With one chamber per well cover the wells with a lid.
Wrap the plate with aluminum foil and place the plate on a rocking platform set to gentle rotation for 1.5 hours in the dark at room temperature. After the incubation period, wash the tissue sections by transferring them to a fresh well containing one milliliter of cold one XPBS wash for at least 10 minutes on the rocking platform. And repeat the washing three times.
Ensure that the tissue sections do not get dried as they can stick to the sides of the chamber. Fix the sections by transferring the tissue chambers into a fresh well. Of the 24 well plate containing one milliliter of filtered 4%PBS buffered para formaldehyde and incubating on a rocking platform for two hours with gentle rotations.
After washing the sections three additional times, place the stained and fixed sections onto a clean microscopic slide. Using a watercolor soft brush, wipe excess water without touching the section and mount the section using mounting medium. Cover with a microscopic cover slip and allow the slides to dry at room temperature overnight.
In the dark room, use a Nikon A one Eclipse 90 I confocal microscope system for routine scanning and acquisition of images. To start open the NIS elements AR software, choose tetraethyl, rumine, isothiocyanate or trit C and cyan five or SCI five channels from the panel. The excitation emission wavelengths for the Trit sea and sci-fi channels appear by default.
Assign the pseudo color green for the Trit Sea Channel and red for the SCI five channel. Place the slide to be examined on the microscopic stage and focus manually under 100 times magnification. Set the HV to one 10 and the offset to zero click focus and adjust the voltage.
To optimize the lasers click focus scan the section from top to bottom and set the Z level for image acquisition. Then click CH series and acquire the image sequentially. Identify dextro or positive CD four positive T cells appearing as yellow punctate cells based on colocalization of signals generated from both the red and green channels.
Use an Olympus FV 500 BX 60 confocal microscope for easy quantitative enumeration of dextro or positive CD four positive T cells. To begin, open the flu ovu software. Select the dyes S SI three and SCI five from the dropdown menu and click apply to load the respective lasers.
Place the slide to be examined on the microscopic stage and focus it manually under low magnification click focus while the SI three laser is on and focus the inflammatory focus. Change the objective to 100 times magnification. Set the file size to five 12 over five 12 from the dropdown menu, click focus and adjust the values for PMT gain and offset to optimize the laser separately for S SI three and sci five, click Z stage and then click focus while both the lasers are on.
Set the thickness of the Z stage by clicking the up and down arrows. Set the Z interval to two micrometers. Click stop and choose.
Seek 1, 2, 3. Then click X, y, Z and start acquiring Z series images for the selected area within the inflammatory focus, save the Z serial images as a VI files. To save image files, select the image from the Z serial image and then save as TIFF format.
Always save multi TIFF images. First to protect data, the next step is to examine three paired sections that have been stained with both sets of dex trimmers. First, take the Z serial images by scanning the slides in a horizontal vertical up horizontal vertical down movement so that the repetition of Z series within the same focus is avoided.
Once Z serial images are captured, name the files by identifying the slide numbers name of the sections and the number of the Z serial images taken. Use image J software to count dextro positive CD four positive T cells in relation to the total number of CD four positive T cells. Select the type of counter in image J check show all and start counting by clicking the center of each cell and continuing to count in different Z serial images by using side arrows.
After counting all the CD four T cells, choose another type of counter and count the dextro positive CD four positive T cells. Click the results tab to get the total number of cells counted with the two different counters. Detection of myelin protio lipid protein or PLP specific CD four T cells was performed using freshly cut cerebral sections derived from EAE mice and stained with cocktails containing anti CD four and PLP 1 39 to 1 51 Dexters or control TMEV 70 to 86 Dexters LSCM.
Analysis of co stain cerebral sections shows cells positive for PLP 1 39 to 1 51 dex trimmers in red and cells positive for the CD four antibody in green, whereas the double positive cells appeared in yellow. The co stain cells showed punctate dots around the periphery. Such a feature was absent in section stained with the control TMEV 70 to 86.
Dex trimmers detection of cardiac myosin specific CD four T cells by in situ staining with cardiac myosin heavy chain alpha 3 34 to 3 52 Dexters was also performed. Analysis of these images by LSCM showed the presence of cells positive for MYHC 3 34 to 3 52 dexters in red cells positive for CD four in green and cells bound with both Dexter and CD four as yellow punctate cells. The background staining for control RNAs 43 to 56 Dexters was negligible Once master, this technique can be done in about eight to 10 hours as opposed to three days with other published protocols if it is performed properly.
While attempting this processor, it's important to remember to prepare the reagents ahead of time and follow the steps systematically. After watching this video, you should have a good understanding of how to section and stain the tissues for enumeration of C-reactive CD four T cells in the target targets.
