Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus (KSHV)
September 14th, 2010
Host cell factors play a critical role in the establishment and maintenance of Kaposi's sarcoma (KS). We outline methods to identify host cell factors altered in KSHV-infected DMVEC cells, and in KS tumor tissue. Cellular genes altered by virus will serve as potential target(s) for novel therapeutics.
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.
View the full transcript and gain access to thousands of scientific videos
Overview
This article discusses the dual labeling of Kaposi's sarcoma tumor tissue using immunohistochemistry to identify viral and host cell proteins. The methodology involves exposing antigens, applying primary antibodies, and developing color for visualization. The findings aim to enhance understanding of host cell factors in Kaposi's sarcoma.
Key Study Components
Area of Science
- Neuroscience
- Oncology
- Immunology
Background
- Kaposi's sarcoma is associated with KSHV infection.
- Host cell factors are crucial for KS development.
- Identifying altered cellular genes can lead to new therapies.
- Immunohistochemistry is a key technique for studying tumor tissues.
Purpose of Study
- To demonstrate the dual labeling technique for tumor tissue.
- To identify host cell factors altered by KSHV infection.
- To provide insights for potential therapeutic targets.
Methods Used
- Heat exposure to reveal antigens.
- Application of biotin-conjugated primary antibodies.
- Use of strep din enzyme conjugate.
- Color development using enzyme substrate.
Main Results
- Successful localization of viral and host cell proteins.
- Immunohistochemical staining provided clear visualization.
- Identified potential host cell factors for therapeutic targeting.
- Enhanced understanding of KS tumor biology.
Conclusions
- Dual labeling is effective for studying KS tumor tissue.
- Host cell factors play a significant role in KS.
- Findings may lead to novel therapeutic strategies.
Frequently Asked Questions
Executive Industry Relevance
Identifying host cell gene expression changes induced by Kaposi's sarcoma-associated herpesvirus (KSHV) supports target validation in oncology drug discovery. This approach enables mechanistic de-risking by clarifying viral-host interactions that drive tumor progression. The method provides predictive confidence for prioritizing therapeutic targets in vascular endothelial and B-cell malignancies.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Interrogates therapeutic hypotheses by linking KSHV infection to dysregulated host genes in dermal microvascular endothelial cells and tumor tissue.
- Operational Value: Enables biological de-risking through confirmation of transcriptional changes via qRT-PCR and protein expression via immunoblot and immunofluorescence.
- Scientific Value: Supports predictive confidence by validating in vitro findings in archival Kaposi's sarcoma tumor tissue using dual-labeled immunohistochemistry and tissue microarrays.
Screening & Assay Development
- Scientific Value: Prepares validated biological systems (DMVEC and KS tumor tissue) for downstream screening of compounds targeting host-viral interactions.
- Operational Value: Standardizes assay outputs through microarray analysis, qRT-PCR, and dual-labeled immunohistochemistry for reproducible quantification of dysregulated genes.
- Scientific Value: Highlights screening readiness by providing quantitative, multi-modal readouts (transcriptional, protein, spatial localization) for reliable compound evaluation.
Translational & Preclinical Research
- Scientific Value: Demonstrates disease relevance by connecting in vitro gene expression changes to KS tumor tissue microenvironment.
- Operational Value: Ensures translational continuity from discovery through preclinical validation using archival tissue and tissue microarrays.
- Scientific Value: Supports risk-adjusted advancement decisions by identifying host factors with potential as prognostic markers or therapeutic targets.
Pipeline & Workflow Integration
The method integrates into the discovery continuum from target validation to preclinical validation, supporting hypothesis testing and biomarker alignment in vascular tumorigenesis.
- Discovery Biology: Supports hypothesis testing by identifying host cell gene expression programs altered after KSHV infection in endothelial cells.
- Screening: Describes assay readiness through microarray analysis and qRT-PCR for quantitative measurement of transcriptional changes.
- Analytics: Highlights multi-omic readouts (transcriptional via qRT-PCR, protein via immunoblot/immunofluorescence, spatial via dual-labeled IHC) that enable comparison of infected versus control conditions.
- Translational Research: Connects method to preclinical continuity via validation of in vitro findings in archival KS tumor tissue using tissue microarrays.
- Enterprise Reuse: Frames the method as a reusable capability for studying viral oncogenesis in other herpesvirus-associated malignancies.
Operational & Enterprise Impact
- Scientific Value: Predictive confidence, target validation, reduction of mechanistic ambiguity in viral oncogenesis.
- Operational Value: Standardization, reproducibility, and scalability of dual-labeled immunohistochemistry and qRT-PCR workflows.
- Strategic Value: Better go/no-go decisions, capital efficiency, and reduced late-stage biological risk in oncology pipelines.
- Portfolio Impact: Risk-adjusted prioritization and advancement decisions based on host-factor dysregulation in KS tumor models.
Implementation Considerations
- Requires expertise in immunohistochemistry, tissue sectioning, and antigen retrieval techniques.
- Dependent on access to formalin-fixed paraffin-embedded KS tumor tissue and tissue microarray infrastructure.
- Necessitates standardization of antibody conjugation, blocking, and washing protocols across laboratories.
- Involves adaptation considerations for different host proteins and viral antigens in dual-labeling experiments.
- Includes practical limitations such as tissue heterogeneity and antigen accessibility in archival samples.
