Histopathology and in situ Hybridisation
The Histopathology/ISH core facility at the Cambridge Research Institute is run with a central theme of high throughput and automation. We offer a full range of histological techniques, immunohistochemistry, in situ hybridisation, laser capture microdissection as well as automated slide digitisation and analysis for the scientists within the CRI.
Histology
The facility offers the capability to process tissues and cell lines into frozen, paraffin or resin formats. In addition to the standard Haematoxylin and Eosin (H&E) staining, we are able to perform any special stains, such as Masson's Trichrome or Periodic-Acid-Schiff. Sectioning of tissue blocks in any of the above formats can be done by facility staff or researchers using two cryomicrotomes and five specialised paraffin microtomes, one of which is enabled for resin sectioning.
We have a Beecher manual tissue arrayer with coring needles ranging from 0.6-3 mm in diameter for constructing tissue microarrays (TMAs).
Immunohistochemistry (IHC)
All antibodies are optimised for automated immunohistochemistry to ensure reproducibility and throughput, although manual methods can still be employed. The facility has two 30-slide Vision Biosystems BondMax autostainers that will complete a fully automated IHC run, including deparaffinisation and antigen retrieval, within four hours. To ensure that the facility employs the best techniques, new methods of fluorescent staining are being investigated.
In Situ Hybridisation (ISH)
The facility currently performs radioactive ISH using S35-labelled riboprobes as a first step, as this provides the greatest sensitivity for this technique. Facility staff are able to provide advice on the design of the probes as well as performing all ISH on the tissue. Non-radioactive fluorescent and DIG-labelled methods are being trialled in the facility with the aim to transfer ISH onto automated staining platforms in the future.
Laser capture microdissection:
The Zeiss P.A.L.M. system is being used within the facility. This instrument is capable of laser excising either single cells or larger tumour areas from tissue sections and placing the cells of interest into a protease-free tube for protein, RNA or DNA analysis extraction and subsequent analysis by the researcher or other core facility.
Digitisation and Analysis
As part of the high throughput theme of the facility, we run two fully automated scanning systems for the digitisation and analysis of tissue. Firstly, the Applied Imaging Ariol SL-50 can be used for the image capture of tissue sections and particularly TMAs with up to a 60x objective in either brightfield or fluorescence. In addition, this has an additional four processor cluster for high throughput batch analysis. Secondly, the Aperio system is used for the fast automated scanning of H&E stained slides and IHC slides, where analysis is not required. Each automated slide-scanning system is connected to 15TB of data storage space.
It is the aim of the facility to provide the best histopathological methods in a high-throughput environment for CRI scientists. Further technical developments to improve efficiency or sensitivity of the methodology will be investigated.
The Histopathology/ISH facility is run by Will Howat (will.howat@cancer.org.uk).
