Minimum Information Specification For In Situ Hybridization and Immunohistochemistry Experiments (MISFISHIE)

There is now a MISFISHIE Working Group within the MGED Society which will provide a permanent home to this developing standard. We welcome all feedback from the community; information about how to join the discussion list can be found at the MISFISHIE Working Group website.

Revised: 2004-12-19

This specification details the minimum information that should be provided when publishing, making public, or exchanging results from visual interpretation-based tissue gene expression localization experiments such as in situ hybridization, immunohistochemistry, reporter construct genetic experiments (GFP/green fluorescent protein, beta-galactosidase), 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.

Modeled after the well-received MIAME (Minimum Information About a Microarray Experiment) specification for microarray experiments, this specification only describes the types of information that should be provided. This specification does not dictate a specific format for reporting the information. We expect to develop a data model based on the concepts of MAGE-OM (MicroArray Gene Expression Object Model) and on the MAGEstk (MicroArray Gene Expression Software Tool Kit) in the near future. It is this model and the associated XML-based markup language that will provide the recommended data format for archiving or transferring such data. Separation of the minimum information specification and the data format is important because the data format should both allow for unlimited additional information as well as some degree of partial information for optimum usefulness.

This is a draft version intended to stimulate discussion. It has not yet been finalized. However, a manuscript is in progress, and we highly encourage anyone interested in being part of this development effort to contact Eric Deutsch or the MISFISHIE discussion list for a copy of the manuscript or with suggestions, comments, or questions.

The following checklist provides a guideline for insuring that data is compliant to the specification.

The MISFISHIE Checklist

Experiment Design:

• Experiment Description: Short summary of the goals of the experiment.
• Assay Type(s): e.g., immunohistochemistry, in situ hybridization, GFP, etc.
• Experiment Design Type: For example, is it a comparison of normal vs. diseased tissue, of multiple tissue specimens of similar type, of multiple probes/antibodies applied to the same tissue, etc.? See MGED Ontology ExperimentDesignType as a basis for categorizing the design type.
• ExperimentalFactors: the parameters or conditions that are tested, such as probe/antibody, disease state, genetic variation, structural unit, age, etc.
• The total number of hybridizations/stains performed in the experiment: a hybridizations/stain is defined as an assay of a single tissue. Thus, an immunostain of a section of a tissue microarray consisting of a 10 x 10 array of different tissues counts as 100 immunostains. If replicates or reruns are a component of the experimental design, provide details that should include number of replicates per tissue, per antibody, per probe, etc., as relevant.
• URL of any websites or database accession numbers that are related to the experiment (if available).
• Contact information for communicating with the experimenter(s).

BioMaterials (specimens) used, and Treatments (section or mount preparation):

• The origin of the biological specimens. Information required includes detailsof the organism (species, strain, genotype, sex, age, developmental stage), the physiologic state of the organism, i.e. normal versus disease, relevant exogenous factors, i.e. treatment, special diet, and the provider of the specimen. All information critical for other researchers to be able to reproduce the biomaterials as closely as possible must be provided. This information is not limited to the above examples. Referencing an established ontology for the terms you use is highly encouraged. The rationale: The location of a population of cells within a specimen is important to know since location may correlate with gene expression. Differential gene expression may be consequent to either tissue handling or to tumor biology.
• The manner of preparation of the specimens for the study. Information required includes the nature of the samples, i.e. whole tissue, tissue sections, thickness of sections, whole cells, or sections of cells, manner in which the specimens were prepared for the experiments, i.e. fixation with type of fixative and duration of fixation versus fresh, non-fixed, non-frozen specimens, versus frozen specimens, sections mounted on slides versus floating in reagents, nature of the slides on which sections were mounted, and the protocols used. Details of how the specimens were stored until use should be provided. For example, if frozen samples are used, provide information regarding storage temperature and duration of storage. Referencing previously published protocols by PubMed ID is permissible if the protocols were appropriately detailed and were followed exactly. If the specimens are on glass slides, details should include section thickness and special characteristics of the slides type of slides, i.e. coating and/or whether the slides are charged slides.

Reporter (probe or antibody) information:

• Unambiguous genomic identification of the reporter:
  • At minimum, the gene identifier and the reference database containing the identifier.
  • If available, the full sequence of the probe, or clone identifiers of the antibody.
  • Since such genomic information may not be available for all antibodies, as much detail that potentially identifies the gene product(s) that are being studied should be provided.
• Protocol for how the reporters were designed and produced or the source from which they were obtained. For GFP-like experiments, the promoter sequence should be specified as the reporter.
  • For reporters purchased from a company, the company name and catalogue number must be provided, as well as the web site that provides details of the specifications, if available. In addition, key aspects in the specifications should be repeated since catalogue numbers and company literature may become unavailable in the future.
  • For a custom made antibody, the putative antigen and references to studies that characterize the sensitivity and specificity of the antibody in tissue immunostains should be provided.
• Additional attributes of the reporter.
  • For antibodies, include the type of primary antibody (monoclonal vs. polyclonal), the immunoglobulin isotype, and the organism in which the antibody was generated.
  • For RNA probes, provide: vector name, cloning site and direction of a cDNA clone, type of labeling NTPs, in vitro transcription (IVT) templates (plasmid template linearized with a restriction enzyme or PCR template with a pair of primers), promoter for IVT labeling reaction.

Staining protocols and parameters:

• Number of detectable reporters (e.g., more than one for multiple-dye confocal fluorescence microscopy) on the hybridization or stain plus specific details about the detection method:
  • Detection reagent used (e.g., fluorescent, enzyme-substrate, gold particles, etc.)
  • Source of the detection system plus sufficient details to obtain or reproduce the reaction product.
• The protocol and conditions used to produce the hybridization or immunostain. This should include the mounting onto the slide/substrate and subsequent treatments of the section, i.e. immunohistochemical stain protocol, including parameters such as buffer, temperature, postwash conditions, etc.). Also include:
  • What steps, if any, were taken to decrease non-specific reaction product. Such steps for immunoperoxidase experiments might include preincubation of the specimen preparation with (a) an albumin solution to block non-specific binding of protein and (b) a peroxide solution to decrease or abolish reaction product catalyzed by endogenous peroxidase.
  • Provide details of any antigen or gene product retrieval method that was used, if any. Such steps for immunohistochemical experiments could include incubation of sections for a specified time in a specified buffer subjected to microwave heating for a specified time.
• Protocols for the assay controls. Information should include the nature of negative tissue controls and negative Reporter controls. Optional specificity Reporter controls, such as competitive inhibition of reaction with either purified protein or peptide for immunohistochemical studies, should be provided.

Imaging data and parameters:

• The images. Any popular file format is acceptable; TIFF or JPEG is preferred.
• Image acquisition parameters:
  • Detection method by which hybridization or staining is observed (for each channel, e.g. fluorescent wavelength, etc., if multiple probes or antibodies are used):
  • Image scale or total instrumental magnification:
  • Image acquisition protocol:
  • Imaging hardware and software used:
  • Image analysis and/or editing software used (if relevant):

Image Characterizations:

The type of characterization that is recorded for such data can vary significantly depending on the experimental design. The following guidelines specify a minimum set of characterization features. Additional characterization of the images as required by the experimental design should also be provided.

• List ontology entries (including reference to ontology, terms, accession numbers) (or provide term and definition if sufficient detail cannot be found in an existing ontology) for each structural unit used for classification. Structural units will be a type of: organ, tissue, cell, etc.
• Define (or choose from an ontology) the staining intensity scale. For example, a three-point scale (of none, equivocal, or intense) is recommended for evaluating CD immunostains. Each gradation of intensity in the scale that is used should be properly defined.
• For each structural unit in each slide (or, in each image), provide quantitative measurements or estimates of:
  • List ontology entries (including reference to ontology, terms, accession numbers) (or provide term and definition if sufficient detail cannot be found in an existing ontology) for each structural unit used for classification. Structural units will be a type of: organ, tissue, cell, subcellular component, etc.
  • Choose from the MGED Ontology the staining intensity scale. For example, a three-point scale of absent, equivocal, or present might be appropriate for evaluating immunohistochemistry stains. However, any scale that the investigators feel is appropriate may be used as long as each gradation of intensity in the scale is defined in a manner that an independent investigator can apply the same set of characterization criteria.
  • For each structural unit in each slide (or, in each image), provide quantitative measurements or estimates of:
    • Staining intensity level or optionally, the fraction of the structural unit population exhibiting each intensity level.
    • Other optional annotations/characterizations of the structural unit, e.g., feature density, qualitative characteristics or spatial distribution of the structural unit or staining. Use of referenced ontology terms is encouraged.
    Both positive and negative measurements of staining relevant to the experiment should be reported.
    For example,

    	Luminal epithelial cell: present
    	Basal epithelial cell: absent
    	etc.
       or:
    	Luminal epithelial cell: 90% present, 10% equivocal, 0% absent
    	Basal epithelial cell: 0% present, 20% equivocal, 80% absent
    	etc.
    

• Protocol for the characterization. Information about the basic technique for characterizing assays should be included, e.g. how many observers performed the characterizations, assessment of inter-observer variability, whether the characterizations were performed from the images themselves or visually through instrument, any exceptions or assumptions made which characterizing the data, etc.

Example datasets

• Example 1: CD49a and CD90 in Human Prostate Tissue
• Example 2: Zebrafish whole-mount in situ hybridizations
• Example 3: Murine developing urogenital tract in situ hybridizations
• More examples needed!