Histology

The Histology facility underpins the research activities of a large number of both basic and translational research groups. It allows the adoption of tissue-based experimental approaches to all research programmes. The unit’s remit is to offer a full range of both routine and advanced histological services for oncology research.

The facility routinely processes, embeds and cuts numerous different tissue types in addition to organotypic assays, spheroids, agar plugs and cell pellets. Vibratome sections from fresh tissue (50–250μm) are also routinely prepared. Cryostat sections are also routinely cut.

Routine equipment within the facility includes a Leica ASP6025 Tissue Processor and embedding station, a Leica Autostainer XL and a Thermo Cryostar NX70 cryostat. The unit also houses the Leica BondMax and BondRX immunohistochemistry platforms together with two Ventana Discovery units, which allow for both exceptional standardisation/quality and ultimate flexibility allowing sophisticated multiplexed labelling techniques to be incorporated into research programs. This ultimately allows more information about the relationships between cells from different lineages on a single tissue sample and the gene expression information in the context of tissue/cell morphology to be analysed. The unit also houses the KOS Microwave Multifunctional Tissue Processor. This is ideally suited as a complement to our existing capabilities, where the multitasking nature of the instrument can enhance and accelerate many of the typical applications encountered. It allows for rapid tissue processing, bone marrow processing and decalcification, special stains, antigen retrieval, fixation and gross hardening.

Fixation, processing and microtomy

No one fixative is ideal, and as result, the unit provides a selection of the most commonly used fixatives, together with advice for correct choice. Specimens are processed on the automated Shandon Excelsior tissue processor using various schedules depending upon the size and nature of the tissue. Paraffin sections may be stained for morphological examination using the Leica autostainer XL, or unstained for protein extraction, immunohistochemical analysis or in situ hybridisation.

Cryotomy

Processing methods can have a deleterious effect on tissue constituents, and as a result, in some circumstances frozen sections may be the preferred method. Advice on all aspects of frozen tissue preparation and post fixation is available. The preparation of fixed cryoprotected samples is also routinely performed.

Special stains

Special stains allow for the demonstration of a variety of tissue constituents. Common requests include lipid, carbohydrate and cytoplasmic granule staining, together with various single and trichrome techniques for connective tissues and extracellular substances. Methods for specialised haematoxylin, nucleic acid and bone preparations are also available.

Immunohistochemistry

Immunohistochemistry is a technique employed by many groups within the Institute. The unit houses the i6000 automated IHC platform and several epitope retrieval stations available for use.

Advice and support in all areas such as choice of fixation, epitope retrieval, method design / deployment and visualization is available.
Optimisation of novel antibodies, multiple chromogenic and fluorescent labelling studies, troubleshooting and interpretation of results are commonly undertaken.

Multiplexing

Cancer research now demands tools that reveal multi-parameter cell level information in intact tissue sections, a need that is not adequately served by traditional single plex immunohistochemistry (IHC). To address this need, multiplex chromogenic and fluorescence immunohistochemistry and in situ hybridisation are now being used both manually and on the automated BondRX platform in the core facility. This allows the simultaneous detection of multiple proteins, DNA and RNA targets at a cellular and sub-cellular level in FFPE tissue sections, retaining spatial arrangement and tissue architectural context, lost with other methods such as flow cytometry.

In Situ Hybridisation

This is an established, available technique. Advice on section preparation, fixation and pre-treatments, plus manipulation of the probe hybridization, stringency washing and probe detection to overcome the unique problems associated with in situ hybridisation is available. Optimisation of novel probes and troubleshooting are often undertaken.

Laser Capture Microdissection

The facility also houses the Leica LMD6000 laser capture microdissection system which, in conjunction with an integrated scanning stage and automated cell recognition facility, provides a powerful tool for the microscopic identification and isolation of pure populations of high quality cells for downstream analysis. In addition the Qiagen Qiacube allows for large scale automated nucleic acid extraction with or without macrodissection.

Although paraffin sections afford better morphological preservation, frozen sections give excellent recovery of both DNA and RNA. Cell identification can be either by morphological analysis or based on immunophenotype.

Tissue Microarrays

Tissue microarrays allow for high throughput analysis and standardisation of methodologies, whilst also preserving rare samples. Both immunohistochemical and molecular detection techniques can be performed on TMA slides.

Tumour-specific, control and custom arrays are routinely constructed. Up to 400 cores can be transplanted to the recipient block.

The facility also has the Beecher manual tissue arrayer (MTA-1) and the automated system (ATA-27). Numerous high quality, pathologically representative arrays have been constructed.

Manchester Cancer Research Centre Biobank

The Histology Facility also houses the Manchester Cancer Ressearch Centre Biobank, an initiative established to facilitate an organised, systematic and comprehensive collection of high quality samples from cancer patients across five different Trusts in the Manchester area.

Developments in genomics and proteomics have highlighted the need for large numbers of tissue and blood samples to be made available to facilitate translational research within the Manchester Cancer Research Centre.