Fixation, Processing and Embedding
We use 10% neutral buffered formalin as our universal fixative. For routine samples we recommend that tissues be fixed in 10% neutral buffered formalin for 24-48 hours and then transferred to 70% ethanol until tissues are processed.
We have two automated tissue processors, and use several processing cycles to handle tissue of varying sizes. Many of our samples require hand processing. These include mouse embryos and pups as well as some thymi. We will custom-process difficult tissue when requested, such as pancreas, mouse heads, and adipose tissue. We decalcify as necessary using Krajian Decalcifying solution, containing formic acid, distributed by J. T. Baker.
We embed tissues using standard universal orientation; however, we will customize our embedding per the requester’s instructions.
Microtomy and Cryosectioning
We routinely section most of our paraffin-embedded tissues at 4 μm unless otherwise specified. Most of our sections are put onto silanized coated slides for better adhesion. We also prepare sections for DNA isolation and slides for in situ hybridization and laser capture microdissection.
We have two cryotomes for cutting frozen tissue samples for routine histology, immunohistochemistry, special stains, laser capture microdissection, and in situ hybridization.
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
Our stainer is a Thermo Varistain Gemini. We use a Gill-2 hematoxylin and an alcoholic eosin-Y from Thermo to produce our hematoxylin and eosin stains. Our staining is very consistent. Every stained slide must be quality controlled before it leaves our laboratory.
Trichrome (three color) staining of the heart
Mammary gland stained with carmine red
Heart and aorta stained with Oil Red O
In addition to routine histological staining, we also perform special staining such as trichrome staining for distinguishing cells from connective tissues, Oil Red O for staining lipds and fats, and carmine red, often used for staining glycogen and other polysaccharides.
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.
We will harvest and fix any tissue using gross techniques or a stereoscope as required for very small organs such as prostate lobes, trigeminal nervesa, etc.
Whole Mount Services
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 routinely stains formalin-fixed, paraffin-embedded tissue sections with a standard three-step colorimetric immunohistochemical detection system. We also use a newer polymer-based two-step peroxidase system when possible. This method helps avoid background staining of endogenous biotin. We also have a super-sensitive tyramide-based signal amplification system we use to enhance immunohistochemical staining when needed. When antigens cannot be detected on formalin-fixed tissues, we can develop protocols for frozen tissue sections and cultured cells adhered to glass slides. Our experienced and knowledgeable ASCP-certified Immunohistochemist is also highly trained in developing new protocols for antibodies as needed for an investigator’s research.
We have incorporated an automated immunostainer into our service to help meet the increasing demand for our services as well as to better standardize commonly used immunohistochemical stains. We have optimized automated protocols for several antibodies that we routinely run on the Biocare intelliPATH Immunostainer. A partial list of automated protocols includes:
- 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. We have made a major effort to expand the number of antigens that we can assay in our core. Presently, we have developed assays to detect well over 500 antibodies. Some of the newest assays detect phosphorylated antibodies (p-AKT, p-STAT-3, p-Rb, p-S6 and p-mTOR), markers of UV damage (CPD and 6-4 PPs), and human cells/tumors grown in mice.
Please contact our staff to inquire about new protocol development. We will be glad to assist you with your immunohistochemical needs.
Chief, Histology Laboratory
Lab phone: 512-237-9309
Office phone: 512-237-9489
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