Immunostaining

Immunostaining

TACMASS offers immunohistochemical staining on formalin fixed, paraffin embedded (FFPE) tissues and cell lines, and on frozen tissues and cultured cells. Chromogenic and fluorescent techniques are available.

Immunohistochemistry (IHC)

TACMASS performs chromogenic immunohistochemistry (viewed with light microscopy) on an automated staining platform. We use the Discovery XT Immunostainer and the associated, validated reagents for antigen retrieval, staining and detection designed for this instrument (Ventana Medical Systems, Inc., Tucson, AZ). The instrument can stain fixed or frozen tissue sections by IHC. It is also equipped to perform In Situ Hybridizaition (ISH).

Primary antibodies used for IHC

TACMASS has validated more than 100 antibodies on the Discovery XT.

Validation includes the process of optimizing conditions for antigen retrieval from formalin-fixed, paraffin-embedded (FFPE) tissues and cell lines on our autostainer. Optimized conditions may vary slightly for human tissues, research animal (mainly mouse) tissues or cell lines prepared in FFPE blocks. Validation also includes optimizing conditions for staining and detection for each primary antibody. The use of an automated staining platform allows for both run-to-run and intra-run reproducibility for each assay.

The selection of primary antibodies that we have validated to date is investigator driven, and we are continually adding new assays to the list upon request. Primary antibodies can be purchased or acquired from a number of vendors and sources.

We supply many of the primary antibodies that we have validated, but investigators may need to purchase primary antibodies* that we have not yet validated. All other reagents for antigen retrieval and detection are specific for this staining platform and are supplied as part of the IHC assay.

*If you are looking for candidate primary antibodies for IHC, check the vendor datasheet for information and images related to the use of IHC performed on FFPE tissues. 

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Pathologic Analysis and Interpretation

TACMASS provides expertise in histological and pathological interpretation of human and research animal material. The Directors assist researchers with experimental design and the establishment of histological scoring strategies. Dr. Ray Nagle, MD, PhD, is a surgical and experimental pathologist with many years of experimental pathology experience. He is available to consult with investigators to analyze and interpret pathological findings in the overall context of the research project. He also oversees quality control issues for pathology related services that are offered by the core lab.

A critical aspect of interpreting immunohistochemical and other assays offered by the service is the identification and use of the appropriate positive and negative control tissues for each assay. TACMASS works with each investigator to identify and acquire appropriate pathological material and controls for IHC and LCM studies.

Immunofluorescence (IF)

TACMASS performs co-localization of proteins by immunofluorescence staining on either frozen tissue specimens or cell lines cultured on coverslips.

We also have a cryostat and the expertise to cut frozen sections for IF staining. We can also cut frozen sections for you to perform IF staining in your lab.

We perform both single and double staining (two antibodies from different host species). We have available both green 488nm FITC (fluorescein isothiocyanate) and red 568nm labeled secondary antibodies made against a variety of species. We can also co-stain with DAPI to localize the nuclei.

Please contact Doug Cromey for training on the Lecia SP5 confocal microscope at the University of Arizona Cancer Center: dcromey@uacc.arizona.edu

Note: Prior arrangements may be made with Dr. Ray Nagle to analyze IF stains.

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Laser Capture Microdissection

The Arizona Cancer Center offers the use of an Arcturus VeritasTM Laser Capture Microdissection instrument through the TACMASS core facility.

TACMASS has the expertise to assist researchers in harvesting either DNA or RNA from formalin fixed, paraffin embedded (FFPE) tissues, or tissues that have been optimally snap frozen.

TACMASS prepares tissues sections and trains users on the Arcturus VeritasTM Laser Capture Microdissection instrument. Users are responsible for all upstream validation steps*, and for all downstream purification and application steps*. TACMASS will supply the special slides, caps and microcentrifuge tubes that are needed for LCM instrument use to users for a small fee (you can also purchase these directly from Molecular Devices).

Please refer to the Applied Biosystems website for more information about this instrument and its capabilities (Applied Biosystems purchased Arcturus). We recommend that you contact their technical services department to discuss details and feasibility for your particular application. They have validated protocols for various types of applications*, and can assist you with experimental design and technical expertise*.

*Molecular Devices has validated purification and amplification protocols using their kits:

  • PicoPure RNA Isolation Kit #0204
  • RiboAmp RNA Amplification Kit #0201
  • PicoPure DNA Extraction Kit #0103

We also recommend that you consult with Dr. George Watts from the Genomics Shared Service if you will be using this core lab for downstream applications prior to initiating your studies.

*To save yourself money and time when harvesting RNA, DNA or protein (particularly RNA) from either frozen or archival tissue blocks, we recommend that you “validate” the integrity of the RNA on each experimental block prior to laser capture. Validation includes harvesting material from a whole tissue section, without laser capturing, using all of the downstream protocols that you plan to use in your experimental design. Ensure that integrity and quality of the experimental material is adequate, and that all steps other than LCM produce optimal material for your analysis.

Please Contact Us to discuss LCM experimental design.

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Design and Construction of Tissue Microarrays

TACMASS works with investigators to design, construct, stain and interpret Tissue MicroArrays (TMAs).  Tissue cores from multiple donor formalin-fixed, paraffin-embedded tissue blocks are re-embedded into a recipient FFPE block.  Cores are 1.5 mm in diameter, with a maximum of 48 cores incorporated into each TMA.   An H&E from each donor block is marked by a pathologist select the area of interest to core.  Histological and immunohistochemical staining procedures can be performed on thin sections cut from the TMA.

Please Contact Us to discuss design and construction of TMAs.

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Digital Imaging

TACMASS has available two microscopes and digital cameras for imaging chromogenic and immunostained microscopic sections:  an Olympus CellSens DP72 Imaging system (this is the camera (in the lab), and a Nikkon Labophot-2 microscope with Paxcam 3 camera and PAX-it Digital Image Management & Image Analysis system (in Dr. Nagle's office).  Quantitation of immunostained microscopic sections is currently under development.