Research Histology, Pathology and Imaging Core - Histology Laboratory Services
The universal fixative is 10% neutral buffered formalin. 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 processing.
We have two automated tissue processors, and use several processing cycles to handle tissue of varying sizes. Many of our samples require hand processing, such as mouse embryos, some thymi and newborn mouse pups. 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, such as cut surface, surface area, or tubular structures; however, we will customize any embedding as per the requester’s instructions.
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 slides for in situ hybridization, and laser capture microdissection and sections for DNA isolation.
We have 2 cryotomes for cutting frozen tissue samples for routine histology, as well as immunohistochemistry, special stains, laser capture microdissection, and in situ hybridization.
We will harvest and fix any tissue using gross techniques or a stereoscope for the very small organs such as prostate lobes, trigeminal nerves, etc.
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, and every stained slide must be quality controlled before it leaves our laboratory.
Tunel, 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.
Whole-mounted mammary glands allow easy evaluation of phenotypic changes.
Whole-mounted prostate, unlike prostate sections, gives a complete overview of the tubular structure of this organ.
The Histology Laboratory routinely stains formalin-fixed, paraffin-embedded tissue sections with a standard three-step colorimetric immunohistochemical detection system. We also use the newer polymer-based two-step peroxidase systems when we can to avoid endogenous biotin background staining. At times, we use the super-sensitive tyramide-based amplification system to enhance immunohistochemical staining. When antigens cannot be detected on formalin-fixed tissues, we also develop protocols for frozen tissue sections and cultured cells adhered to glass slides.
Our experienced and knowledgeable ASCP-certified Histology Immunohistochemist is also highly trained in developing new protocols for antibodies needed for an investigator’s research.
We have incorporated an automated immunostainer into the service to help with the increased number of stains and slides and to further standardize the more routine immunohistochemical stains. Several antibodies have been optimized, automated, and are 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
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 can be assayed by our core, and presently we have assays developed to detect well over 350 antibodies. Some of the newest assays are for detecting phosphorylated antibodies (p-AKT, p-STAT-3, p-Rb, p-S6 and p-mTOR) and for markers of UV damage (CPD and 6-4 PPs), as well as detecting 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.