The histopathology/ISH core facility offers a variety of histological techniques including immunohistochemistry, in situ hybridisation, laser capture microdissection, as well as slide scanning and image analysis to CRUK CI scientists.
The core provides routine histological processing, tissue sectioning, as well as immunohistochemistry and in situ hybridisation. Histological services include the capability to process animal tissues and cell lines into frozen or paraffin formats. In addition to the standard hematoxylin and eosin (H&E) staining, the facility can perform special stains, such as Masson’s Trichrome, Periodic-Acid-Schiff, Reticulin and Oil Red O. The Facility constructs tissue microarrays for Institute researchers.
The Facility has two Leica Bond III autostainers, and one Bond Rx research instrument for performing fully automated immunohistochemistry (IHC) as well as a further Bond Rx research instrument, dedicated to automating in situ Hybridisation (ISH) assays. These 30-slide systems are highly standardised and will complete an IHC run, including deparaffinisation and antigen retrieval, within 2–4 hours. We routinely run 2,000 slides per month, which demonstrates the robustness of these systems. The Facility has 400 antibodies optimised to date, and has also worked up tyramine amplification for weak staining and the TUNEL assay for apoptosis/necrosis.
The Facility is also able to perform dual IHC/immunofluorescent (IF) staining as well as co-detection of both RNA or DNA and proteins.
The Facility uses RNAscope (single/duplex chromogenic and 3/4 plex fluorescence) and BaseScope assays from Bio-Techne for detection of RNA in tissue sections. This technology, performed automatically on the Leica Bond Rx, will produce up to 30 ISH slides overnight, thus increasing the facility’s throughput and capacity. We offer miRNA ISH using LNA probes in combination with OPAL fluorophores and also have a DNA FISH service for human and mouse pan-centromeres, XY chromosomes, or other commercially available probes of your choice.
The Facility also houses a Leica LMD7000 for Laser Capture Microdissection.
The majority of slides produced in the facility are digitised automatically, with 354,000 digitised to date. This is enabled through:
A standalone microscope capable of brightfield, fluorescence and darkfield imaging with Zen (from Zeiss) image capture software.
Zeiss AxioScan Z1
Images both fluorescence and brightfield up to 40x magnification with a resolution of 0.11µM/pixel. This fully automated high throughput scanner can hold 100 slides and is able to resolve up to 5 fluorophores in a single image.
Leica Aperio AT2 system
Automated brightfield scanning is carried out on the Aperio AT2 at both 20x or 40x magnification with resolutions of 0.5 and 0.25uM/pixel respectively. This fast-high throughput scanner can hold 400 slides and can scan a slide in approximately 2 minutes.
Cirdan Xplore Software
Cirdan Xplore software is a web-based image viewing platform used for sharing images with external collaborators, slide scoring and TMA segementation.
Indica Labs Halo
Halo image analysis software is available for our internal users, and can be provided either as a service or bookable equipment. We have a number of modules for both brightfield and fluorescent applications. The modules include positive pixel quantification, multiplex ISH spot counts as well as tissue-based analysis and TMA segmentation. Halo software is compatible with many image types including those produced by both of our scanners, tiffs and the images created on the Fluidigm Hyperion (Imaging Mass Cytometer) within our flow cytometry core facility.