⚠ NOTE
By contributing to the knowledge-base you are agreeing that the provided data be made available under a Creative Commons Attribution 4.0 International license and that you will abide by the code of conduct Contributor Covenant.

The knowledge-base was made possible by researchers like you, see who they are and our thoughts on overcoming the fear of sharing.

To contribute your knowledge you need the following:

  1. Open Researcher and Contributor ID (ORCID), your unique scientific identifier. If you do not have an ORCID, register for it here.
  2. A GitHub account. If you do not have an account, follow these instructions to sign up for one.

This knowledge-base follows the standard GitHub fork and pull request, triangular workflow.

One time setup

  1. If you are comfortable working with git and GitHub from the command-line, install git. Otherwise, download the GitHub Desktop application and read the installation and configuration help.
  2. Fork the IBEX Imaging Community repository and clone it to a local directory (GitHub Desktop instructions).
  3. If you are adding reagent information (including reproducing results of existing configurations), contributing a protocol or sharing a new video tutorial, add your information to the “creators” section in the .zenodo.json file (This is a hidden file. To see it on windows, open file-explorer and select View->Show->Hidden items). Note: Additions should maintain alphabetical order based on last name and be inserted between the first and last two entries (these three entries remain in their fixed locations in the list).

Adding information to the knowledge-base

Before starting

Create a new local git branch based off the main branch (GitHub Desktop instructions).

Modifying the publications.bib file:

Simply add an entry to the file using the appropriate bibtex entry type.

To give credit where credit is due, we ask that you provide the complete list of authors and do not truncate it, even if it is long.

Modifying a markdown file in the docs directory:

Simply edit the relevant file.

Some markdown files are automatically generated (e.g. index.md, reagent_resources.md), do not edit these files. At the top of auto-generated files you will find a line reading: “Do NOT edit this file. It is automatically generated from…”, edit those source files.

Modifying protocols.csv:

Add your protocol details to this csv file, title, URL which links to the protocol and the protocol details (a short abstract describing it). Do not use non-ASCII characters in the title or description. Represent them using standard ASCII, instead of α write alpha.

Modifying videos.csv:

Add your video details to this csv file, title, URL which links to the video, the video details (a short abstract describing it) and the date. We use separate columns for year, month, day so that we do not need to remember an order, mm/dd/yy or dd/mm/yy and tools like MS Excel will not modify entries in an attempt to help us.

Modifying reagent_resources.csv:

Do not use non-ASCII characters such as ™ or α (either remove or represent using standard ASCII “alpha”). For explanation of the column contents see the reagent metadata section.

Note: If using excel or google docs to edit the file, you need to be careful if your preferences include automatic data conversion, as this may change the file content incorrectly. In our case, a conjugate such as 9E10 will be converted to a number, 90000000000. In excel, disable the “Automatic Data Conversion” before opening the file. In google docs open an empty spreadsheet and then import the csv file. During the import processes you will first select the delimiter, comma in our case. You will then need to specify the column type as “text” for all columns.

Adding new line:

  1. Add a line to the reagent_resources.csv.
  2. Create corresponding supporting material sub-directory using the target_conjugate as the directory name and add a markdown file with your ORCID as the file prefix (see existing files for reference). Supporting material file has a fixed layout, see supporting_template.md.
    Note: When creating the target_conjugate sub-directory you will need to replace the following characters with an underscore: space, tab, “/”, “", “{“, “}”, “[”, “]”, “(“, “)”, “<”, “>”, “:”, “&”. Consequently, supporting material for the target conjugate pair of “Chicken IgY (H&L)”, “FITC” is found in a directory named “Chicken_IgY__H_L__FITC”.
  3. Optionally add small (<300KB) image files (jpg) and refer to them in the supporting material file. Please format your images as 72dpi, 24bit color, 1200x1200px. Write caption identifying validated reagent. Use colorblind safe colors (e.g. cyan, red; magenta, green, blue; no green and red). Include scale bar. Use zoom that allows fine details to be seen. See an example here. Add the file information to the image_resources.csv file found in the data directory.
    For general information about image quality and content for use in supporting scientific claims see:

Adding information to existing line:

We only accept up to 5 ORCID additions in the Agree or Disagree columns. This means that, the original contributor’s work was replicated by up to 4 people from other laboratories or refuted by up to 5 people from other laboratories.

  1. Add your ORCID to the Agree or Disagree column on the appropriate line in the reagent_resources.csv file. Use a semicolon ; to separate from the previous ones (e.g. ORCID1; ORCID2; ORCID3).
  2. If after adding your vote:
    • The number of ORCIDs in the Disagree column is greater than those in the Agree column:
    1. Change the content of the Recommend column from “Yes” to “No” or the other way round and swap the ORCIDs between the Agree and Disagree columns.
    2. Modify all the supporting material markdown files associated with this row, changing the ResultXXX and RecommendationXXX sections.
  3. Add a markdown file with your ORCID to the existing sub-directory. The easiest option is to copy the contents of one of the markdown files already in this directory and modify it.
  4. Optionally add small image files (jpg) and refer to them in the supporting material file. Please format your images as 72dpi, 24bit color, 1200x1200px. Write caption identifying validated reagent with orthogonal marker (see below). Use colorblind safe colors (Cyan, red; magenta, green, blue; no green and red). Include scale bar. Use zoom that allows fine details to be seen.

After work is completed

After completing the work, update the public knowledge-base. This can be done using git on the command-line or using GitHub desktop:

  1. Commit the changes (GitHub Desktop instructions).
  2. Push the commit to GitHub (GitHub Desktop instructions).
  3. Create a pull-request on GitHub (GitHub Desktop instructions).

Reagent Metadata

Adapted from the Affinity Reagent Working Group of the Human Biomolecular Atlas Program (HuBMAP) antibody metadata standards.

For meanings of acronyms and other terminology see glossary.

Field Description Required format Examples
Uniprot Accession Number Identifies the target protein (see uniprot.org). If UniProt ID cannot be found for the antibody, please list NA, e.g., secondary antibodies, nuclear dyes, and labeling kits. Alphanumeric; for multiclonal (pan-) antibodies, list IDs for all targeted proteins as a comma delimited list. A2BC19, P12345, Q9BZS1
Reagent Type Broadly defines a reagent used for multiplexed imaging. Capitalize first letter of each word. Primary Antibody, Secondary Antibody, Zenon Labeling Kit, Nuclear Dye
Target Name Provides a common name for the target protein being detected by assay. Please spell out Greek letters (alpha, beta, gamma, delta, etc). Commonly used name or protein abbreviation. CD20, ICAM, Somatostatin, Alpha smooth muscle actin, TCR gamma delta
Target Species Identifies the species reactivity of a primary antibody. Capitalize first letter of each word. Human, Mouse, Rhesus, African Green
Host Organism Identifies the species in which the antibody was raised. Capitalize first letter. Rabbit, Donkey, Mouse
Isotype Describes the antibody isotype. Please write out any symbols. Do not include kappa or lambda light chains. IgG, IgG1, IgG2a
Clonality Provides the clone ID (if monoclonal) or identifies the antibody as polyclonal. List clone as provided by the manufacturer. If polyclonal, enter Polyclonal L26, EPR5386, Polyclonal
Vendor Provides information on the source of the antibody. Vendor name. Cell Signaling Technology, Abcam, BioLegend. Please carefully check spelling of vendor names and cross reference with entries in the vendors_and_urls csv file before submitting new entries.
Catalog Number Provides catalog number from vendor for the source of the antibody. If the antibody is a custom conjugate, then please list the catalog number for the unconjugated antibody. Vendor catalog number. Ab9566, sc-20060, C6198, 300401 (Unconjugated)
Conjugate Specifies fluorophore conjugated to antibody or no fluorophore (Unconjugated). For fluorophores, please use industry standard abbreviations (e.g., AF for Alexa Fluorophore™). Please do not include trademarks because they do not render properly in .csv files. AF647, PE, iF594, eF570, Unconjugated (if no fluorophore)
RRID Allows for universal identification of an antibody (search by catalog number in the RRID Portal). If there is no entry, please register your antibody or list NA. Several vendors list RRIDs on their website (BioLegend, Thermo Fisher Scientific, BD Biosciences). AB_###### AB_793620, AB_10124480, NA
Availability Lists whether a reagent is commercially available (stock) or a custom reagent. If the reagent is custom, then please provide the catalog details for the conjugation kit in the Notes section of the supporting material file. Capitalize first letter. Stock or Custom
Method Describes imaging configuration used for reagent evaluation. Multiplexed 2D Imaging refers to single cycle imaging with fluorescently conjugated antibodies. IBEX2D Manual refers to thin (<20 um) tissue sections that are manually imaged as outlined in [Radtke et al. 2020]. IBEX 2D Automated refers to thin (<20 um) tissue sections that are imaged with ARIA as outlined in [Radtke et al. 2022]. Please see Glossary for definitions. Method, tissue thickness 2D (<20 um) or 3D (>300 um), Manual, Automated IBEX2D Manual, IBEX2D Automatic, Multiplexed 2D Imaging
Tissue Preservation Preservation technique used. If fixative other than formalin, indicate the percentage of fixative used (e.g., 1% or 4%). Use a common abbreviation format (e.g., FFPE for formalin fixed paraffin embedded). FFPE, 1:4 Cytofix/Cytoperm Fixed Frozen (Method described in [Radtke et al. 2022])
Target Tissue Lists the tissue used for imaging. Capitalize first letter of each word. Jejunum, Liver, Small Intestine, Tonsil
Tissue State Provides details about the state of the tissue such as type of cancer, infectious agent, or immunization status. Include NA if the tissue status is unknown or not critical for the performance of an antibody. Capitalize first letter of each word. Follicular Lymphoma, Metastatic, Senescent, Mycobacterium Tuberculosis Infected, NA
Detergent Describes the detergent, if any, used in blocking, staining, and wash buffers. This is critical for antibody performance. Please include NA if no detergent is used. Percentage and type of detergent with first letter capitalized in detergent name. 0.3% Triton-X-100, 0.1% Saponin, NA
Antigen Retrieval Conditions Describes antigen retrieval conditions used for FFPE tissues. List NA for fixed frozen tissues. pH of the buffer, temperature, and time used for antigen retrieval. pH 6 for 40 minutes at 95C (AR6 Akoya Biosciences AR600250ML), pH 6 (ER1 buffer AR9961) for 30 minutes on Leica Bond
Dye Inactivation Conditions Details the concentration of LiBH4 and time required to extinguish signal. If the fluorophore does not bleach (Hoechst), include NA. Concentration of LiBH4 with time and “+ light” if dye inactivated in the presence of light (BV421, BV510 conjugates). 0.5 mg/ml LiBH4 10 minutes continuous exchange with automated protocol, 1 mg/ml LiBH4 15 minutes, 1 mg/ml LiBH4 30 minutes (FITC), 1 mg/ml LiBH4 for more than 120 minutes (AF594, eF615), 1 mg/ml LiBH4 15 minutes + light (BV421, BV510), NA (Hoechst)
Recommend Details whether you recommend (Yes) or do not recommend (No) a reagent for the tissue type and experimental workflow (tissue preservation method, antigen retrieval conditions, detergent, and imaging method). Elaborate on recommendation using the Notes section in the supporting material file. Yes, No Yes, No
Agree Identifies the individual who validated the reagent using the same experimental conditions (same tissue source, tissue preservation method, detergent, and imaging method). Please register for an ORCID. Limited to 5 individuals. ####-####-####-#### (the last digit may be X) 0000-0000-0000-0000
Disagree Identifies the individual who was unable to validate the antibody using the same experimental conditions (same tissue source, tissue preservation method, detergent, and imaging method). Please register for an ORCID. Limited to 5 individuals. ####-####-####-#### (the last digit may be X) 0000-0000-0000-0000
Contributor Identifies the individual who contributed the reagent. Please register for an ORCID. ####-####-####-#### (the last digit may be X) 0000-0000-0000-0000

Glossary

Several of the definitions here are adapted from the glossary defined in the SOP written by the Affinity Reagent Working Group of the Human Biomolecular Atlas Program (HuBMAP).

Term Definition
Clearing-enhanced 3D (Ce3D) Tissue clearing method that allows volumetric imaging of thick tissue sections (>200 microns to 3 mm) using direct immunolabeling with fluorescently conjugated antibodies [Li et al. 2019, Li et al. 2017].
Ce3D-IBEX Multiplexed 3D imaging method that combines Ce3D and IBEX for visualization of up to 15 markers in a single tissue section using conventional fluorophores and commercially available instruments. The method is currently under development and led by Dr. Hiroshi Ichise, Armando Javier Arroyo-Mejias, and colleagues in the Germain laboratory at the NIH [Arroyo-Mejias et al. 2022, Germain et al. 2022].
Cell DIVE The Cell DIVE multiplexed imaging solution is an integrated system for imaging 60+ biomarkers from one tissue sample at the single-cell level. The tissue-preserving staining and dye inactivation workflow and advanced image processing enables a more comprehensive understanding of the tissue microenvironment than has been available with traditional chromogenic or fluorescence-based imaging [Gerdes et al. 2013].
Cell DIVE-IBEX Method employing the IBEX dye inactivation protocol with compatible fluorophores on the Cell DIVE platform. This method allows imaging of thin, 2D (5-10 micron) tissue sections.
Conjugate Fluorescent dye, enzyme, or hapten covalently attached to a primary or secondary antibody that is used for downstream detection of the antibody bound to its target in/on a cell.
Detergent Reagent included in blocking, staining, and wash buffers to reduce non-specific labeling, enhance reagent spreading, and permeabilize cells for nuclear labels. Examples include saponin, Tween-20, and Triton-X-100.
Fixed Frozen Method of tissue preservation where samples are placed in fixative (e.g., formalin), cryoprotected with sucrose, frozen in molds with optimal cutting temperature (OCT) media, and then stored at -80C. The 1:4 Cytofix/Cytoperm Fixed Frozen method refers to the protocol outlined in the original IBEX manuscripts [Radtke et al. 2020, Radtke et al. 2022].
Formalin Fixed Paraffin Embedded (FFPE) Most common form of tissue preservation where tissues are saturated with formalin and then embedded in a block of paraffin wax. Samples are stored at room temperature.
Fresh Frozen (FF) Method of tissue preservation where samples are frozen at ultralow temperature without fixative (fresh), immobilized in cryosafe media (e.g., OCT, carboxymethyl cellulose), and stored at -80C.
IBEX2D Manual IBEX configuration that relies on manual immunolabeling and dye inactivation as initially described in [Radtke et al. 2020] and detailed further in Box 2 of [Radtke et al. 2022]. This method uses thin, 2D (5-30 micron) tissue sections. The resulting image can be either a single plane or a small volume.
IBEX2D Automated IBEX configuration that uses a fluidics device to deliver antibodies and dye inactivation solution directly to the sample with minimal user intervention. This method uses thin, 2D (5-10 micron) tissue sections and was described in a detailed protocol [Radtke et al. 2022]. The resulting image is a single plane.
Iterative Bleaching Extends Multiplexity (IBEX) An open source, community supported iterative immunolabeling and chemical bleaching method for high-content imaging of diverse tissues [Radtke et al. 2020, Radtke et al. 2022].
Multiplexed 2D Imaging Standard, single cycle (non-iterative) imaging method applied to thin, 2D (5-30 micron) tissue sections. Prior to inclusion in an IBEX imaging panel, all antibodies are first validated using this method. Most antibodies that are recommended for Multiplexed 2D Imaging are compatible with IBEX if conjugated to a LiBH4-sensitive dye. Antibodies that are not recommended for this method are not suitable for IBEX imaging. The resulting image can be either a single plane or a small volume.
Opal-plex Method that utilizes signal amplification with Opal dyes and the IBEX dye inactivation protocol to perform multiplexed imaging in heavily fixed tissues and/or detect low abundance epitopes in conventionally fixed tissues. This method was developed by Dr. Emily Speranza and colleagues in the Germain laboratory at the NIH [Radtke et al. 2020].