MISFISHIE - Minimum Information Specification For an In Situ Hybridisation or Immunohistochemistry Experiment

This specification details the minimum amount of information that should be provided when publishing, making public, or exchanging results from visual interpretation-based tissue gene expression localisation experiments such as in situ hybridisation, immunohistochemistry, in situ reporter genetic experiments, etc.

Compliance to this standard is expected to provide researchers at other labs enough information to reproduce the experiment and/or to fully evaluate the data upon which results are based.

It was modelled after the MIAME (Minimum Information About a Microarray Experiment) specification for microarray experiments.

For more information read the Nature Biotechnology MISFISHIE paper.

There are 6 basic parts to these types of experiments that should be described to a minimum level to allow others to repeat or interpret your experiment:

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 1. Broad Information about the experiment  

The information about an experiment usually refers to multiple assays and includes:

  • Contact information for communicating with the experimenters.
  • Assay Type (ISH, IHC, ISR, lectin binding etc)
  • Experimental Design Type (e.g. testing multiple probes in one tissue? or testing 'normal' vs. different mutants?)
  • Experimental Factors (the parameters or conditions that are tested, such as probe or antibody, disease state, genetic variation, structural unit, age, etc.)
  • Number of assays in the experiment.

 

  
2. The Detection Reagent  

This means the probe or antibody. Define them as completely as possible.

  • What gene(s) or protein(s) is being detected by this reagent? For mouse, ideally use the MGI identifier for the gene (e.g. Fgf8 = MGI:99604)
  • For nucleic acid probes, ideally provide the full sequence. If denoting a sequence using co-ordinates against a GenBank sequence accession ID, ensure you use the relevant version (e.g. NM_12345.1 or NM_12345.2, not simply NM_12345 as the length of the sequence can vary substantially between versions). Template clone names such as IMAGE:IDs can be used additionally. Is the template cDNA or genomic DNA? How is the the probe labelled (e.g. digoxigenin, S35)
  • For antibodies, ideally provide the antigen sequence that was used to generate the antibody. If obtained from a third party, give the supplier name, catalogue number and batch number. Is the antibody monoconal or polyclonal? What species was the antibody raised in? Is the primary antibody labelled (and if so, what is the label)?
  • For in situ knock-in reporters, this information is included in the specimen description and need not be included here (e.g. the description of the reporter allele).
  • Manner in which the detection reagent was prepared (e.g. cRNA probe synthesis procedures). Referencing previously published protocols is permissible if the protocols are appropriately detailed and were strictly followed.

 

  
3. The Specimen  
  • Species, strain and mutant allele information. For mouse use MGI strain and allele information.
  • Sex, Developmental Stage, Genotype, Phenotype.
  • Specimen Type (e.g. wholemount or section)
  • Manner in which the specimen was prepared (e.g. fixation and embedding reagents and procedures). Referencing previously published protocols is permissible if the protocols are appropriately detailed and were strictly followed.

 

  
4. The Staining Procedure  

How the detection reagent and the specimen were mixed together and how the detection reagent was visualised.

The protocol used to produce the hybridisation or immunostain. Referencing previously published protocols is permissible if the protocols are appropriately detailed and were strictly followed.

  • What steps, if any, were taken to decrease nonspecific reaction product?
  • Any secondary, tertiary, quaternary sandwiching reagents used.
  • The final label used for visualisation (e.g. radioactive isotope, fluors, enzymes such as Peroxidase or Alkaline Phosphatase).
  • The detection method/reagents used to detect the final label (e.g. autoradiography, fluorescence, DAB or NBT/BCIP substrates)
  • Information about controls: the nature of both positive and negative controls (or state if controls were not performed). The same level of detail for the tissue controls should be reported as for the cells or tissues that are being studied.

 

  

5. Data images

  
  • Digital images for each assay in the study, digitally available for download without charge.
  • Images should be of sufficient resolution to allow independent characterisation and provided in a standard file format (for example, JPEG, PNG, GIF, TIFF).
  • Detection method by which hybridization or staining is observed (for example, for each channel, fluorescence excitation and emission wavelengths if more than one reporter is used).
  • If the detection method is the same for all images, it need only be mentioned once.
  • Images for the controls are optional.

 

  

6. Characterisation of the image

  

A description of what is observed in the image. i.e the expression pattern. For EMAGE, the simplest way is to create a text annotation using the anatomical terms in the EMAP anatomy ontology. EMAGE staff will also provide a spatial annotation for you in a collaborative manner.