Tyramide Signal Amplification (TSA) is a powerful technique used in immunohistochemistry to enhance the sensitivity and specificity of antigen detection in tissue samples. It also enables simultaneous detection of multiple distinct proteins (multiplex immunofluorescence, mIF) in a single tissue sample.

    Horseradish peroxidase (HRP) acts as a catalyst in the TSA process, facilitating the conversion of fluorophore-conjugated tyramide substrates into highly reactive radicals that covalently bind to nearby tyrosine residues on or near the protein of interest. Primary and secondary antibodies can be stripped away from the section without compromising the fluorescent signal, allowing multiple staining rounds even with primary antibodies raised in a single species.

    The service

    We provide a 7-color (6 markers + DAPI as counterstain) multiplex immunofluorescence (mIF) staining service in human or mouse tissue samples. The staining is performed by using automated Leica BOND Rx research stainer at the Histology core facility, enabling processing of up to 30 slides at once.

    We have a limited collection of primary antibodies available to use in your marker panel. Any research-specific antibodies can be also included in the panel if they meet the specific requirements. Please see more information in the Markers tab.

    If you are interested in using the mIF staining service, please check more details about the workflow in the Sample submission tab, and don’t hesitate to ask directly from tissue-mif@utu.fi. In addition to the mIF service, Leica BOND Rx can be used for many other technologies (e.g. RNA in situ hybridization) by request. Please see more details in the Instruments section.

    Samples
    • Formalin-fixed, paraffin-embedded (FFPE) tissue
    • 4-6 µm sections on a regular adhesive (positively charged) microscope slides (no rounded or clipped corners)
    • Human or mouse tissue specimens
      Research-specific markers
    • Primary antibodies produced in mouse or rabbit
    • Prior to mIF staining, a routine IHC (e.g., chromogenic DAB staining) is highly recommended in order to find the optimal antibody concentration and staining condition for each marker in the target tissue
    We have a limited collection of primary antibodies available to use in your marker panel. Any research-specific antibodies can be also included in the panel if they meet the specific requirements (see more in the Sample Requirements tab). Markers can be paired with any of the six fluorophores to create the optimal mIF-panel case-by-case. There are some things to consider when designing the panel, for instance tissue autofluorescence, the stability and abundance of the target protein, and its cellular localization.

    >>Human-specific primary antibodies

    >>Mouse-specific primary antibodies  

    Sample submission and project workflow

    1. Initial consultation – send an email to: tissue-mif@utu.fi:
    Describe your plan shortly:
    • Tissue type
    • Markers of interest
    • Research objective – what and how you want to analyse from the staining

     Let's create a staining plan together (e.g. short meeting face-to-face or online).

    1. Fill in a Tissue multiplex IF staining project request in OpenIris (for billing information).
    2. Fill in a Tissue multiplex IF staining sample submission form in OpenIris.
    • Details for the staining based on the discussed plan
    • Fill in a new sample submission form for each new staining request
    1. Sample (sectioning and) delivery:
    If you have already sectioned samples on slides, bring them to Histocore sample drop-in at Medisiina D, 5th floor (reception table marked with the sign Incoming samples). Alternatively, order tissue sectioning service directly from the Histology core facility (>>detailed instructions).
    1. Staining:
    Staining will be performed by the Histocore personnel using the Leica BOND Rx research stainer.
    1. Imaging:
    You will be notified when the slides are ready for pick-up (same place as for sample delivery but reception table marked with the sign Outgoing samples). The TSA-fluorescent dyes are relatively stable. However, if you are unable to image them immediately, please store the stained slides at +4 degrees covered from light.

    Things to take into consideration

    Recommended steps to take when starting a new multiplex-IF staining project (first: send e-mail to tissue-mif@utu.fi).
    1. Optional, but highly recommended:
    If you are using your own research-specific antibodies, routine IHC with Leica BOND Rx is recommended to test the optimal staining conditions (e.g. primary antibody concentration, antigen retrieval buffer of choice).
    1. Optional:
    Routine IHC can be performed with Leica BOND Rx to test the staining service primary antibodies in your tissue of interest (list of tested tissues and representative images can be found in an excel sheet in the Markers tab).
    1. Optional: Single IF-staining of each marker
    • IF-staining should resemble the chromogenic staining (if done in a parallel tissue sample). Issues with tissue autofluorescence and antibody concentration can be also evaluated when comparing IF and chromogenic staining side by side (isotype control can be stained in parallel to a single IF).
    • In addition to staining order, selection of the fluorescent dye can have an effect on the staining results. For example, choosing fluorescent dyes with far-red and near-infrared emission can improve signal to noise ratio in tissues with high autofluorescence.
    • Images from the single IF-staining can be used as a reference if performing spectral unmixing for image analysis.
    1. Optimization test: Multiplex-IF staining (e.g. one test slide + one IgG control slide)
    • Repeated HIER (heat-induced epitope retrieval) treatment for antibody stripping in multiplex-IF staining can increase (most antigens get more retrieved by repeated HIER) or decrease (some protein epitopes can be degraded by repeated HIER) the signal depending on the target of interest. Single IF-staining should resemble the target signal in the multiplex-IF staining. For example, an issue with signal loss could be avoided by changing the staining order.

    Options for mIF imaging in Turku campus area

    • Leica Thunder Widefield microscope (up to 6 markers + DAPI, located at BioCity 5th floor)
    • Leica STELLARIS 8 FALCON FLIM Confocal microscope (6 markers or more + DAPI, located at BioCity 5th floor)
    • Nikon Eclipse Ti2-E Widefield microscope (limited to 5 markers + DAPI, located at BioCity 5th floor)
    • Pannoramic Midi Fluorescence slide scanner (limited to 3 markers + DAPI, located at Medisiina C2)
    New! Fluorescence scanner coming in 2025 For more information about imaging, please contact microscopy@bioscience.fi