Fixation, Processing and Embedding
The core uses 10% neutral buffered formalin as its universal fixative. For routine samples, tissues should be fixed in 10% neutral buffered formalin for 24-48 hours before being transferred to 70% ethanol for processing.
The core typically uses an automated tissue processor and protocols designed to handle tissues of varying sizes. The core can also hand-process samples such as mouse embryos and pups, and will custom-process special or difficult tissue as requested. Samples are decalcified, if necessary, with Krajian Decalcifying solution containing formic acid ( J. T. Baker).
Tissues are embedded using standard universal orientation unless the researcher has other specific requirements.
Microtomy and Cryosectioning
Core personnel routinely sections paraffin-embedded tissues at 4 μm, unless otherwise specified. Most sections are placed on charged slides for better adhesion. Sections can also be prepared for DNA isolation, in situ hybridization, and laser capture microdissection.
The core staff can prepare frozen tissues from frozen tissue samples for routine histology, immunohistochemistry, special staining, and laser capture microdissection.
The primary core stainer is a Thermo Varistain Gemini. For hematoxylin and eosin stains, core workers use a Gill-2 hematoxylin and an alcoholic eosin-Y (Thermo), which produce consistent results. Every stained slide is quality controlled before it leaves the core laboratory.
Hematoxylin and eosin (H&E) stained heart and blood cells
H&E stained epithelial cells magnified 20x
H&E stained epithelial cells magnified 40x
H&E stained kidney cells
In addition to routine histological staining, the core can also use special stains such as trichrome for distinguishing connective tissues, Oil Red O for lipids and fats, Carmine Red for glycogen and other polysaccharides, and numerous other staining protocols.
Trichrome (three color) staining of the heart
Mammary gland stained with carmine red
Heart and aorta stained with Oil Red O
Active Caspase-3 and cleaved Lamin A assays can be used to detect the number of cells in a given tissue sample that are undergoing programmed cell death.
Tissue Sampling and Whole Mount Services
Core personnel will harvest and fix any tissue using gross techniques or a stereoscope, as required. Whole-mount techniques allow observation of structures within a larger context. For example, whole-mounted prostate, unlike prostate sections, gives a complete overview of the tubular structure of this organ. Likewise, whole-mounted mammary glands allow easy evaluation of phenotypic changes within the entire gland.
The Histology Laboratory performs both automated and manual immunostaining and reoutinely stains formalin-fixed, paraffin-embedded tissue sections with a standard three-step colorimetric immunohistochemical detection system or a polymer-based two-step peroxidase system, which helps avoid enodgenous biotin staining. The core also uses a sensitive, tyramide-based signal amplification system for enhancing immunohistochemical staining when needed. When antigens cannot be detected on formalin-fixed tissues. The core can develop protocols for frozen tissue sections and cultured cells adhered to glass slides. When antigens cannot be detected on formalin-fixed tissues, core staff can develop new protocols using frozen tissues and other fixation methods.
The core's automated immunostainer has helped meet the increasing demand for the core's services and helped standardize commonly used immunohistochemical stains. Optimized, automated protocols for several antibodies routinely run on the Biocare intelliPATH Immunostainer include:
- Proliferation markers: 5-bromo-2'deoxyuridine (BrdU), Ki-67, PCNA
- Differentiation markers: Keratins 1, 5, 6, 8, 10, 13 and 14, S100 A8, S100 A9
- Cell cycle related proteins: Cyclin D1, E2F1, p27, p21
- Oncogenes and tumor suppressor genes: Rb, p53
- Tagged proteins: FLAG, B-gal
Manual Immunostaining and New Protocol Development
A machine can never fully replace a skilled technician. Antibodies of limited quantity and those requiring special attention will always be stained by hand. Core personnel have made a major effort to expand the number of antigens that can be assayed in the core. Protocols have been developed for over 500 antibodies. Some of the newest assays detect phosphorylated proteins (p-AKT, p-STAT-3, p-Rb, p-S6 and p-mTOR), markers of UV damage (CPD and 6-4 PPs), and markers for human cells/tumors grown in mice.
Please contact the core regarding new protocol development for your research needs.
Jimi Lynn Young, M.S., HT (ASCP)
Research Laboratory Manager
Lab phone: 512-237-9309
The Virginia Harris Cockrell Cancer Research Center
at the University of Texas MD Anderson Cancer Center, Science Park, Department of Epigenetics and Molecular Carcinogenesis
Mailing Address: P.O. Box 389, Smithville, Texas 78957
Physical Address: 1808 Park Road 1C, Smithville,Texas 78957