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Project: Submission Spreadsheet

Title: IHC Example Entries

 

Introduction

The purpose of this document is to detail the requirements for filling out entries in the spreadsheet designed for submission to the GUDMAP database.  An Immunohistochemistry (IHC) entry encompasses data relating to embryos of one sex, and one Theiler stage, but can include data from multiple embryos.

Conceptually, the spreadsheet is divided into multiple sections, which include the following:

Submission ID

(Column 0)

Person Details

(Columns 1 to 17)

Probe Details I

(Columns 20 to 36)

Probe Details II

(Columns 84 to 90)

Specimen Details

(Columns 37 to 47)

Images

(Columns 48 to 50)

Annotation I

(Columns 51 to 53)

Annotation II

(Columns 77 to 83)

Related Data

(Columns 54 to 56)

Acknowledgements & References

(Columns 57 to 71)

Tissue

(Column 72)

Non-Wildtype Alleles

(Columns 96 to 105)

Transgenic Reporter Alleles

(Columns 91 to 95)

Results Notes

(Column 106)

 

IMPORTANT:  Only alpha-numeric characters (A-Z, 0-9) will be accepted in both the image directory and the image filenames.  The only exception to this is an underscore [ _ ] and the dot [ . ] before the file extension.

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Submission ID (Column 0)
We would like a unique ID for each row in the spreadsheet (a submission is one row in the spreadsheet).
We would prefer that you use the Temporary GUDMAP ID generated, for each submission, by the Online Annotation Tool.

This takes the following format:

999999n

For submissions that have not been annotated using the Online Annotation Tool, please construct the identifier using the following format:

PI’s initials_DDMMYY_n

(The Principal Investigator’s two initials followed by the date and an incrementing integer.)

Essential fields are marked in red.

Submission ID

 

 
Submission ID Help

 

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Person details (Columns 1 to 17)
We would like you to complete this section as much as possible.  Essential fields are marked in red.

 

  Essential fields are marked in red.
Person Details Help

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Probe details I (Columns 20 to 36)

These columns contain details to uniquely identify the antibody you have used. In column 20 the submitters are asked to specify an antibody name. Columns 23-25 describe the epitope by use of a SwissPROT ID. Further details can be provided in the Probe Additional Notes (column 27). The columns should be filled in such a way that an investigator could repeat the experiment.

Essential fields are marked in red.

Probe Name

The antibody name is entered here.

 

Accession Number

The SwissProt ID of the epitope is entered here.

Probe Extends From and To

The start and end coordinates of the epitope are entered here.

Gene Symbol

Enter a MGI-recognised gene symbol here.

Antibody Details

 

Probe details II (Columns 84 to 90)
Towards the end of the spreadsheet, we have provided seven columns in which you can include more details relating to how the antibody was raised and how the IHC staining was accomplished. The examples below show you how to fill in these columns.

Variant Detected

If the antibody recognises e.g. a splice variant, enter the details here.

Final Label

Enter here either the colourimetric or fluorescent label (e.g. HRP, AP, FITC, TRITC).

Supplier

We would like the following:

  • Company Name
  • Catalogue Number
  • Lot Number

Use the pipe symbol (|) to separate these details. If details are not known please enter 'na'.

Antibody Type

Enter here whether the antibody is monoclonal or polyclonal.

Antibody Chain Subtype

If a monoclonal antibody, enter here the antibody chain subtype if this is known (e.g. IgG1, IgG2a).

Antibody Details

Production methods used to generate the antibody. We would like to know whether the following details are applicable:

  • Hybridoma (monoclonal)
  • Phage Display (monoclonal)
  • Species immunised (polyclonal)
  • Purification method (mono/polyclonal)
  • Ig Isotype (mono/polyclonal)

Use the pipe symbol (|) to separate these details. If details are not known please enter 'na'.

Antibody Species Specificity

Enter here the species with which the antibody is known to cross-react.

Use the pipe symbol (|) to separate species.

More Antibody Details

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Specimen details (Columns 37 to 47)
In this section we would like you to detail the stage, strain, and genotype of the embryo and also the fixation / embedding procedure used. Essential fields are marked in red.

Specimen Stage Value

The Theiler stage of the embryo is entered here.

Other Staging System

Enter here the alternative staging system from the drop-down menu.

dpc - days post-coitum

pnd - postnatal days

sm - sexually mature

Other Staging Value

Enter here the numerical value of the other staging system if appropriate.

For adult specimens, leave this blank.

Specimen Strain

Enter here the specimen strain.

Sex

Enter here either male, female or unknown from the drop-down menu.

Genotype

If the embryo is derived from a mutant or transgenic line, we would like you to state 'non-wild type'.

Mutation

Enter here the MGI ID of the mutated allele.

Specimen Preparation

Indicate whether the specimen is a wholemount, a section, or a wholemount subsequently sectioned.

Fixation Method

Enter here the fixative.

Embedding

Enter here the embedding medium.

Discussion Notes

Enter here additional notes describing in more detail the IHC experiment.

Specimen Details

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Images (Columns 48 to 50)
In this section, we would like you to enter the filenames of the supporting image files plus a short note for each image.

Image File

Enter here the filenames of the supporting image files. Ensure you add the appropriate file extension (.tif, .jpg, etc.)

Use the pipe symbol (|) to separate multiple filenames.

Other Staging System

Enter here a note for each image.

Use the pipe symbol (|) to separate image notes. If you do not wish to supply an image note for an image, please enter 'na'.

Image Details

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Annotation I (Columns 51 to 53)
In this section we would like you to provide ontology ID’s relating to the structures in which genes are expressed.

The examples below detail the correct method of completing High Resolution annotation (section data).

Expression Present

Enter here the GUDMAP ontology IDs of structures that are labeled.

Use the pipe symbol (|) to separate multiple IDs.

Expression Not Detected

Enter here the GUDMAP ontology IDs of structures that are not labeled.

Use the pipe symbol (|) to separate multiple IDs.

Annotation Notes

Entries in the annotation notes should be of the format 'ID=note'.

The IDs can be from either the 'Expression Detected', 'Expression Uncertain' or 'Expression Not Detected' columns.

Use the pipe symbol (|) to separate multiple annotation notes.

Annotation Details

 

Annotation II (Columns 77 to 83)
Following the International Annotation meeting held in Edinburgh during August 2006 there has been requests for the option to annotate anatomical structures with a perceived strength.  It was decided at the meeting that this should be allowed only if a confidence level (1-3) was introduced, and only if the confidence were high (3) would strength be assigned.

Level of Confidence (Column 77)
Enter here the confidence rating of your ability to delineate expression strengths of the annotated structures in this submission.  Confidence is on a scale of 1 to 3, where 1 is low and 3 is high.

For each submission that has a confidence rating of 3, further details can be provided in the Expression Strength columns.

Note that only those structures annotated as Expression Present in Annotation I (and only those) should be assigned an Expression Strength.

Level of Confidence

Enter here the confidence rating of your ability to delineate expression strengths of the annotated structures in this submission.

Confidence is on a scale of 1 to 3, where 1 is low and 3 is high.

Expression Strength Strong

Enter here the GUDMAP ontology IDs of structures that show strong expression.

Use the pipe symbol (|) to separate multiple IDs.

Expression Strength Moderate

Enter here the GUDMAP ontology IDs of structures that show moderate expression.

Use the pipe symbol (|) to separate multiple IDs.

Expression Strength Weak

Enter here the GUDMAP ontology IDs of structures that show weak expression.

Use the pipe symbol (|) to separate multiple IDs.

More on Annotation

 

In addition to the strength of gene expression, we would additionally like to know more about the pattern of gene expression.  This information can be entered into the ‘Expression Pattern’ column (column 81).  An example of how to fill in this column is shown below:

Expression Pattern

Entries in the expression pattern should be of the format: ID=pattern?location&pattern?location where the ampersand (&) precedes all but the first pattern and the question mark (?) precedes all locations. Locations are optional.

Options for patterns include: ubiquitous; graded; regional; spotted; single cell; restricted.

Options for locations include: radial; dorsal/ventral; rostral/caudal; medial/lateral; proximal/distal; deep/surface; adjacent to (EMAP ID).

The IDs referred to can be from either the 'Expression Present' or 'Expression Uncertain' columns. Anatomical structures referenced in the 'Expression Not Detected' column cannot be assigned a pattern.

Use the pipe symbol (|) to separate multiple IDs.

FOR EDITORIAL USE

DO NOT USE

Expression Uncertain

Enter here the GUDMAP ontology IDs of structures of which the expression is uncertain.

Use the pipe symbol (|) to separate multiple IDs.

NOTE: This column is to be left blank if the entry has been assigned a confidence level of 3 in column 77.

Expression Pattern

 

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Related Data (Columns 54 to 56)
In this section we would like to know if the submission is linked to another submission in one of the following databases:

Related Databases

The Related Data columns should be completed as described below:

Related Data Database

Databases to which the data are linked are entered here.

Use the pipe symbol (|) to ensure a one-to-one correspondence between the databases and the Related Data Submission IDs (provided in the following column).

Related Data Submission ID

The submission ID of the related data is entered here.

Both the prefix and the entry ID should be entered.

Use the pipe symbol (|) to separate multiple IDs.

Related Data Type

Enter here the parameter by which the data is related.

Options include: same section; same embryo; same probe; same experiment; control. Use the ampersand (&) symbol to refer to multiple options.

Use the pipe symbol (|) to ensure a one-to-one correspondence between the databases and the Related Data Submission IDs.

Related Data

 

If the data you wish to higlight as related are within the spreadsheet that you are sending, then simply enter the submission ID's (Column 0) of these linked submissions in Column 55.

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Acknowledgements (Columns 57 to 63)
In this section columns are provided to allow the submitter to acknowledge collaborators or companies that have assisted in the generation of the ISH data.

Acknowledgements


References (Columns 64 to 71)
In this section columns are provided to allow the submitter to refer to publications in which the ISH data has been published. 
Rather than completing all the relevant columns, if a Pubmed ID is available for the publication then filling in the first column labelled References PMID shall suffice.

References

 

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Tissue (Column 72)
In this column we would like you to list the tissues you have analysed. Enter terms or EMAP ID’s from the GUDMAP ontology. Use the pipe symbol (|) to separate multiple ID's.

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Non-Wildtype Alleles (Columns 96-105)
Please contact the GUDMAP Editorial Office directly about submitting non-wildtype/mutant allele data.

 

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Transgenic Reporter Alleles (Columns 91-95)
In this section columns are provided to allow the submitter to document details of the transgenic reporter allele(s), if applicable.

Transgenic Allele ID

Enter here the MGI ID for the transgenic reporter allele.

If a MGI ID is unavailable, please enter the prefix and ID of an alternative database (e.g. GENSAT).

Transgenic Allele Name

Enter here the name of the transgenic reporter allele.

Transgenic Reporter

Enter here the type of reporter (e.g. GFP, lacZ, luciferase etc).

Transgenic Reporter Visualisation Method

Enter here the visualisation methods used to detect the reporter. Examples include:

  • anti-GFP Ab
  • transgene fluorescence
  • B-gal+Xgal
  • luciferase+luciferin

 

Transgenic Reporter Notes

Enter here any supplementary notes you feel may be useful in interpreting the transgenic reporter expression (e.g. leaky expression of transgene in lumenal structures).

Transgenic Reporter Allele

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Results Notes (Column 106)

In this section submitters can add additional information about this entry.

Results Notes