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Our Science - TARP Frequently Asked Questions

GENERAL QUESTIONS:

TECHNICAL QUESTIONS ABOUT IMMUNOHISTOCHEMISTRY:

How can I use TMA slides?
 Tissue arrays have wide utility; in theory, anything you can do with a regular section of tissue you can do with a tissue array. Arrays can be used for in situ hybridization (both RNA and DNA), immunofluorescence (including confocal), immunohistochemistry, and in traditional histochemical staining methods. Other methodologies are being developed. The vast majority of slides are used for immunohistochemistry.

How do you store the slides?
 Slides should be stored in a closed container and protected from dust because slide surfaces are tacky. Stored slides should not be stacked directly on top of each other or in direct contact.

Some labs choose to store their slides in zip-lock bags with desiccant in the refrigerator (4ºC), or freezer (-20ºC). Other groups go to other extremes including nitrogen gas, dipping in paraffin or storage under vacuum. These efforts are to prevent slide oxidation, a poorly understood phenomenon that results in decreased immunogenecity of stored sections. This phenomenon is dependent on both tissue processing and the antigen being examined. We try to maintain a fresh stock of slides and use only the highest quality donor blocks.

Where does TARP tissue come from? Can I get more information on the tissue or frozen samples?
 The tissue used to construct the TARP arrays is obtained from the Cooperative Human Tissue Network (CHTN). The tissue is excess surgical resection tissue – in other words, tissue that is left over after the pathologist has taken sections required for diagnosis. The tissue is formalin-fixed and paraffin-embedded. Some history is available from surgical pathology reports, but this information is very limited. We are working to include more clinical data, but age and sex are unknown for more than 10 percent of cases. The tissue is anonymous and delinked, meaning that we cannot obtain more information or additional tissue. The tissue is obtained with the understanding that it will be used to depletion. We make every attempt to use this resource to its fullest. Matched frozen tissue, additional history, or whole sections from the blocks are not available.

How do you design the TARP Arrays?
 The TARP Arrays are designed to maximize the archival tissue that is obtainable for construction of the arrays. We aim to present statistically relevant numbers of tumors that will allow an investigator to screen potential targets. We consider the TARP Arrays a screening tool. They allow investigators to ask simple questions about pattern of expression, differences between tumor types and such. The TARP Arrays are not powered for organ specific research – either in the design of the array, or the annotation that is available.

The prostate tumors are segregated to their own array for two reasons: 1) high demand for prostate tumors 2) it is the only tumor type for which we have matching normal tissue for the bulk of all tumors.

Other tumor types are included based on the availability of tumors in sufficient numbers to warrant inclusion. The newest multitumor arrays are broken into two arrays: T-MTA-2A and T-MTA-2B. Array A contains the more common cancers seen in the US: colon, lung, breast and ovarian cancer. All of these tumors are carcinomas. Array B contains a more complex mix of tumors of different origins, including (but not limited to): melanoma, glioblastoma multiforme (a high grade type of brain tumor), lymphoma (a mix of different histologic subtypes), renal cell carcinoma, carcinoma of the thyroid, and other tumor types when we have sufficient samples available.

Tell me about the Instrumedic tape slides.
 The Instrumedic tape-sectioning method is used to produce the TARP slides for several reasons. The method makes a very durable slide that withstands antigen retrieval quite well. The methodology for making the sections does not require a water bath to float the sections. As a result, there is no loss of sections—each turn of the microtome blade produces a slide. Because the tape is applied to the block and the paraffin section is not floated on water, there is no stretch or distortion in the array, assuring precise alignment of spots on the slide. There are disadvantages, including lumpy histology and difficulties in determining the slide’s quality before staining. The tape-sectioning methodology includes a UV-sensitive acrylic-coated slide and a carrier tape. The slides are sticky because of the acrylic coating that is sometimes on the back of the slides. The only effective way to remove this coating is with a razor blade.

How do you tally the results?
 We use the time-tested method of creating a grid of the array on a sheet of paper and using highlighters in different colors. This was the basis for redesigning TARP2 into units of 25 (5x5)—so a unit could easily be visualized with a 4x objective and did not require spot counting to determine exact location. We mark spots as absent, unreadable, and based on whatever grading schema we are using (positive versus negative [0 to 4+]). Scoring is rapid and provides a permanent record that is easy to track.

How do you image the slide and present the data?
 Capturing images of individual spots is relatively straightforward, but anything more than a 5x5 unit of spots is challenging with a regular microscope. Special microscopes from several manufacturers can scan an entire slide and create an image of the entire array. We usually summarize the data and show representative spots.

TECHNICAL QUESTIONS ABOUT IMMUNOHISTOCHEMISTRY:

What are the recommended protocols for immunohistochemistry?
 We have no absolute protocols for immunohistochemistry. Any basic protocol or immunohistochemistry kit is suitable. Functionally, the slides are nearly identical to regular slides.

We do recommend increasing deparaffinization, hydration and dehydration times at least 25 percent over the standard protocol time, to 7.5–10 minutes per step, because of an altered interaction of water with the adhesive slides. Automated immunostainers can be used with care. Autostainers that use a liquid coverslip technology or a capillary method may not work as effectively with TMAs.

I tried deparaffinizing the slides there is a haze on the slides. Did they deparaffinize?
Relax! The slides are deparaffinized. The slides are made with adhesive slides from Instrumedics . The adhesive on the slides is a long chain acrylic adhesive and it will take on a haze like appearance during the staining process. A routine deparaffinization process will deparaffinize the slides. This haze will be retained throughout the staining process until a coverslip is applied. See: Tell me about the Instrumedic tape slides

What are the recommended protocols for antigen retrieval?
Antigen retrieval is a very passionate subject among those who do immunohistochemistry. The tape sections are quite hardy and withstand antigen retrieval well. Although we do not recommend using a microwave for antigen retrieval with the array slides, it can be done. The TARP Lab uses a rice cooker/vegetable steamer or pressur cooker. Other methods that work include the use of autoclave ovens. The choice of buffer and antigen retrieval time is antigen dependent. We have not noticed appreciable spot loss except under extreme conditions.

My slides have a background haze after staining where there is no tissue. How do I get rid of it?
We have encountered a haze in some staining protocols, including some H&E protocols, and for Masson's trichrome and other stains. This haze is an interaction with the tape adhesive. After staining, soak the slides in 70 percent ethanol for 10–30 minutes, then dehydrate and coverslip. This approach will not alter insoluble chromagens for immunohistochemistry.

What are the recommended protocols for in situ hybridization?
As for immunohistochemistry, we have no special protocols. General hydration recommendations for immunohistochemistry apply. We have used colorimetric, avitin-biotin complex methods with good results for U6, a ubiquitously expressed mRNA, and EBNA1, an EBV transcript. Others have had excellent results with radioactive methods.

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