Histopathology and in situ hybridisation
The Histopathology/ISH core facility offers a variety of histological techniques, immunohistochemistry, in situ hybridisation, laser capture microdissection, as well as automatic slide digitisation and analysis to CRUK CI scientists.
The facility processes, embeds and sections human and animal tissues or cell lines into frozen or paraffin formats and stains these with the standard H&E or special stains, as needed by the researcher to complement their work. We have been increasingly using vibratome sectioning from 50–250 μm in thickness to examine cellular interactions in three dimensions and for cultures of live tissue following sectioning, with great success (Figure 1). We have also added von Kossa for the demonstration of calcium, Feulgen for nuclei and the Gomori trichrome method to our arsenal of special stains.
Figure 1. A three-dimensional reconstruction of a 300 µm vibratome section of pancreatic tumour which has been cultured with a population of immune cells (red), demonstrating the localisation of those cells to the tumour (green) and the surrounding connective tissue (blue).
The immunohistochemistry arm of the facility has continued to provide high throughput services for CRUK CI researchers, with approximately 15,000 slides stained for routine markers. In addition, we have been requested to validate a further 17 antibodies onto the Bond stainers which will become routine markers in the future, taking the total of validated antibodies to more than 400. Following the purchase of the Leica Bond III system at the beginning of the year, we have closed down two of the Bond II systems, and have donated one of these to the University of Cambridge. We have continued to validate dual colour IHC and aim to increase the amount of dual immunofluorescence that we offer.
In situ hybridisation (ISH)
In collaboration with Affymetrix, we have been evaluating an automated version of their manual branched DNA non-radioactive ISH method that was validated last year. This is designed to operate on the Leica Bond Rx that was purchased earlier this year and will allow full automation of the ISH process including deparaffinisation and pretreatment of the tissue sections. This will enable us to run 30 slides overnight, thus increasing the throughput of the facility and increasing the ease of the wuse of ISH to the Institute. In addition, we have continued to run our standard miRNA staining method and fluorescence ISH (FISH) for the Y chromosome and human/mouse centromeric regions. We aim to continue to work on alternative amplification methods for RNA and DNA and the dualling of these techniques with IHC.
Digitisation and analysis
We have digitised 28,000 slides through our three scanners (Leica Ariol SL50, Aperio XT, Zeiss Mirax) this year. We continue to work with increasing numbers of researchers who are using image analysis as part of their work as we offer nuclear, cell surface, cytoplasmic, microvessel density and positive pixel analyses to the scientists in the building, as well as training in tissue recognition analysis software where required. As part of the University of Cambridge, we are in collaboration with other University scientists, including those from the Addenbrooke’s Histopathology Department, the John van Geest Centre for Brain Repair, the Hutchison/MRC Research Centre and the Veterinary School for both digitisation and image analysis.