Multi-plex Immunofluorescence (IF) and Imaging
Use of the Akoya Biosciences Polaris system allows up to 6 epitopes to be assayed in a single tissue section. These can be tailored to your project or selected from off the shelf kits. The same system can be used to determine the localisation of several RNA molecules in a single sample. We can also perform additional H&E and chromogenic IHC staining.
THL have a variety of panels available that are already optimised in mouse and human tissue. Below are combinations of antibodies we have used in the past, which can be translated onto future projects. These may need testing and possibly re-optimising in order to tailor it to your specific tissue and requirements.
For more examples of panels please click the link below.
Due to the nature of the file sizes, we can only store data until the end of each project.
A multiplex panel can consist of up to 6 different antibodies, in addition to DAPI.
You can receive/view your multiplex slides in the following formats:
1. Spectrally mixed whole slide scan -qptiff (Phenochart/QuPath)
2. Spectrally unmixed tiles -tif
3. Spectrally unmixed whole slide scan -tif – (You can view these in HALO)
We will return your slides once they have been processed to the stage you require as above.
- Old tissue is often poorer quality than newer tissue. We recommend re-embedding in fresh wax prior to use.
- Tissue is better preserved in the block than on the slide, for example, historically cut slides often give poorer staining. We recommend sectioning close to the staining date.
- Optimal formalin fixation results in better IHC or multiplex staining.
- There should only be one tissue section per slide.
- Place tissue as centrally as possible on the slide.
- If intending to overlay images for analysis, tissue placement and orientation must be as similar as possible across each slide.
- Thick tissue sections give poorer quality staining. 5-micron sections are recommended.
Please see the FAQ ‘What factors affect the quality and consistency of multiplexed images?’ for further multiplex related considerations.
- Large tissue sections and TMAs show more variation than smaller sections. Sections no larger than 2cmx2cm are recommended.
- We recommend the use of TOMO slides which preserve tissue substantially better than conventional coated slides.
- Epitopes that share the same sub-cellular localisation (e.g.in the same receptor complex) may be prone to steric hindrance. Careful selection of panels is therefore recommended.
Please see the FAQ ‘What factors affect the quality and consistency of brightfield images?’ for further considerations.
- Adipose (fat) rich areas, such as those within breast and pancreas tissue, can show poor staining and are prone to lifting off the slide. TOMO slides are always recommended in these tissues.
- Calcifications, which occur in tissues such as breast and serous tumours, can cause artefactual defects in tissue.
- Tissues rich in endogenous peroxidases such as muscle and liver often show more non-specific staining.