Protocol for MICS experiments

MICS (MACSima™ Imaging Cyclic Staining) is a cyclic immunofluorescence staining method for ultrahigh-content imaging of samples using the MACSima Imaging System. All steps including the immunofluorescence staining are carried out in a fully automated manner by the instrument.

Protocol

  1. Prepare sample(s) for immunofluorescence staining using either FFPE- or PFA-fixation.
  2. Mount the sample(s) onto MACSwell™ Sample Carriers.
  3. Pipet the individual antibody conjugates at their final dilution into their dedicated well positions on a MACSwell Deepwell Plate, as indicated by the MACSima™ Instrument software.
  4. Place the antibody-containing MACSwell Deepwell Plate and the mounted sample(s) into the instrument.
  5. Follow the instructions of the software to start the experiment. The instrument will perform the cyclic staining procedure in a fully automated manner. 
    One MICS cycle comprises the following steps:

    a. 10 min immunostaining at room temperature
    b. Image acquisition
    c. Signal erasure, which is achieved either via photobleaching when using conventional or REAfinity™ Antibodies, or via controlled signal release by addition of REAlease™ Release Reagent when using REAdye_lease™ or REAlease™ Antibodies.

6.  Analyze acquired image data using Qi Tissue image analysis software.

Note: MICS-validated antibodies are also suited for standard immunofluorescence microscopy. The optimal experimental conditions have to be determined by the user.

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