The overall goal of this procedure is to demonstrate the dual labeling of Kaposi's sarcoma tumor tissue by immunohistochemistry for viral and host cell proteins. This is accomplished by first exposing antigens using heat. The second step of the procedure is application of primary antibody conjugated to biotin.
The third step of the procedure is the addition of the strep din enzyme conjugate. The final step of the procedure is the addition of the enzyme substrate to the tissue for color development. Ultimately, results can be obtained that show the localization of both viral and host cell proteins in the tumor tissue through immunohistochemical staining.
This method can answer key questions in the field of pathology, such as identifying infectious agents as well as dysregulated cellular genes in the same specimen. Under a fume hood, place up to eight chem eight slides with five micrometer sections of fixed paraffin embedded tissue in a coplan jar of xylene. Incubate for 10 minutes to de parize and to prepare the slides for dual labeled immunohistochemistry.
Rehydrate the tissue in a grade series of 100%95%and 70%ethanol solutions for five minutes each. Transfer the slides to water for at least 10 minutes before antigen unmasking, also known as antigen retrieval for antigen unmasking. Submerged the slides in a plastic container containing freshly prepared 95 degrees Celsius.
50 millimolar citrate buffer heat in a microwave oven to maintain the boiling at 95 degrees Celsius for 15 minutes. Immediately transfer slides to a coplanar full of distilled water at room temperature. Transfer slides to a clean coplan jar filled with freshly prepared quenching solution.
Incubated room temperature for 30 minutes to quench the endogenous peroxidase. Wash three times in room temperature one XPBS for five minutes each wash. Remove the slides from the PBS wash and shake off the excess covered the tissue with 200 microliters of ice cold blocking solution cover with a strip of paraffin to prevent drying.
Place the slides in a humidified chamber and incubate at room temperature for one hour. Remove the strip of paraffin and shake excess blocking solution off the slide at 50 to 100 microliters of ice cold primary antibody against KSHV Lana diluted in blocking buffer to cover the tissue. Recovered the tissue with a fresh paraffin strip incubate in the humidified chamber for one to two hours.
Wash the slides three times in one XPBS for five minutes. Each wash add 50 to 100 microliters of biotinylated secondary antibody diluted in one XPBS incubate in the humidified chamber at room temperature for 30 to 45 minutes. While the slides are incubating, prepare the peroxidase conjugated streptavidin.
Using the daco animation duet kit, add 10 microliters of solution A to one milliliter of one XPBS in a 1.5 milliliter einor tube and vortex to mix. Add 10 microliters of solution B and vortex again incubate at room temperature for 30 minutes. Wash the slides in one XPB S3 times for five minutes each wash.
As before, add 100 to 200 microliters of horse radish peroxidase conjugated strippin to the slides. Incubate at room temperature in the humidified chamber for 30 to 45 minutes. Again, wash the slides three times in one XPBS for five minutes per wash.
Five minutes before the end of the washing drop one DAB tablet into a 50 milliliter conical tube containing 20 milliliters of one XPBS and 25 microliters of 3%Hydrogen peroxide. Filter the DAB solution through a 0.45 micrometer syringe filter working with one slide at a time. Cover the tissue with one milliliter of DAB solution under brightfield illumination on a light microscope.
Monitor color development for KSHV Lana in infected cells when the desired color development is achieved. Submerge the slides and distilled water for five minutes to stop the reaction for dual labeling. Begin again with antigen unmasking in 95 degrees Celsius citrate buffer as described in the accompanying text and stain.
Using the same procedure, mount the tissue with per mount mounting medium and cover with a cover slip image under brightfield illumination. It is often desirable to send out many individual specimens embedded in paraffin blocks to be punched into, arranged into a tissue microarray for further statistical analysis here. Brown cells positive for the cellular protein pcam one CD 31 indicated with a gray arrow cluster around blood vessels.
Red cells positive for the viral protein. Lana marked with black arrows indicate cells infected with kaposi's sarcoma associated herpes virus. Here is a lower power magnification of brown cells positive for the cellular protein pcam.
One CD 31 indicated with gray arrows in a region around blood vessels. Red cells positive for the viral protein. Lana marked with a black arrow indicate cells infected with kaposi's sarcoma associated herpes virus.
Here is a KS tumor tissue biopsy on a tumor tissue microarray brown cells positive for viral protein. Lana indicates cells infected with kaposi's sarcoma associated herpes virus and show some spindle cell formation. Here is another KS tumor tissue biopsy on a tumor tissue microarray brown cells positive for viral protein.
Lana indicate cells infected with kaposi's sarcoma associated herpes virus with a high degree of spindle cell formation. After watching this video, you should have a good understanding of how to perform dual label immunohistochemistry on formal and fixed paraffin embedded tissue. While performing this experiment, it's very important to watch color development under the microscope and quench the reaction in water.
