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Authors: Liz McDonough, Chrystal Chadwick, Fiona Ginty, Christine Surrette, Anup Sood
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community, GE Research
Summary: This protocol describes the process of validating antibodies (primary/secondary, direct conjugates, and zenon labelled) as per the Cell DIVE™ technology. It also describes the process for determining any antigen effects from the dye inactivation process.

Proper citation: Liz McDonough, Chrystal Chadwick, Fiona Ginty, Christine Surrette, Anup Sood 2020. Cell DIVE™ Platform | Antibody Characterization for Multiplexing. protocols.io https://dx.doi.org/10.17504/protocols.io.bpyxmpxn Copy   

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Authors: Marda Jorgensen
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: Method for sectioning paraffin blocks for staining

Proper citation: Marda Jorgensen 2019. FFPE Tissue Sectioning for Staining. protocols.io https://dx.doi.org/10.17504/protocols.io.8kvhuw6 Copy   

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Authors: Michelle Daniel, Marda Jorgensen
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: The following SOP describes procedures required for the antibody staining of formalin fixed,paraffin-embedded (FFPE) samples. In this SOP, the term “samples” refers to FFPEtissue samples.

Proper citation: Michelle Daniel, Marda Jorgensen 2020. Imaging Mass Cytometry Antibody Staining . protocols.io https://dx.doi.org/10.17504/protocols.io.bfz8jp9w Copy   

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Authors: Anup Sood, Eric Williams, Liz McDonough
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community, GE Research
Summary: ScopeThe purpose of this protocol is to conjugate purified antibodies to Cy dyes. Antibody conjugation involves combining dyes with antibodies to achieve a fluorescently labeled antibody. Each antibody will be labeled with a molar equivalent of dye; the quantitative output of this is deemed D/P. In the initial conjugation step, each antibody will be labeled with differing D/P levels and validated to determine which level is optimal.

Proper citation: Anup Sood, Eric Williams, Liz McDonough 2021. Cell DIVE™ Platform | Ab Conjugation: Initial Conjugation & Scale up Conjugation. protocols.io https://dx.doi.org/10.17504/protocols.io.bp55mq86 Copy   

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Authors: Jamie Allen, Jeff Spraggins, Danielle Gutierrez
Group: VU Biomolecular Multimodal Imaging Center, Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: Scope:To describe the procedure for the lysis, reduction/alkylation, trypsin digestion, and clean-up of tissue. Lysis will cover the lysing of tissue and protein concentration. Acetone precipitation will cover the precipitation of proteins. Digestion will cover the process for digesting 100 µg of protein using the Agilent AssayMap Bravo Robot and Promega Rapid Trypsin/LysC. The clean-up of the cells will cover the desalting process on the Agilent AssayMap Bravo and the subsequent preparation of the samples for LC/MS peptide analysis.Expected Outcome/Data:Cell samples lysed, digested, and desalted for analysis on MS instrument. Samples to be analyzed within one or two days of desalting.

Proper citation: Jamie Allen, Jeff Spraggins, Danielle Gutierrez 2019. Automated, Rapid Preparation of Tissue Sections for Proteomic Analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.67nhhme Copy   

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Authors: Michelle Daniel, Marda Jorgensen
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: This SOP describes the tuning of an Imaging Mass Cytometer (IMC). The tuning processencompasses parameters such as Interface alignment, gas flow rate optimizations, laserpower optimization and detector voltage optimization. All these factors aim at having amachine that performs equally over time.The machine needs to pass specifications listed in this SOP in order to be used for dataacquisition.Machines do not need to be tuned daily but at least twice per week and upon every startup.Additionally, the adjustment of the laser energy is described in this SOP, whichneeds to be performed every 3 weeks.

Proper citation: Michelle Daniel, Marda Jorgensen 2020. Imaging Mass Cytometer Tuning . protocols.io https://dx.doi.org/10.17504/protocols.io.bf2gjqbw Copy   

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Authors: Elizabeth Neumann, Jamie Allen, Heath Patterson, Danielle Gutierrez, Jeff Spraggins
Group: VU Biomolecular Multimodal Imaging Center, Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: Scope: Obtain autofluorescence microscopy images of tissues after IMS analysisExpected Outcome: A brightfield and green autofluorescence microscopy image of the tissue section that enables registration and correlation of different imaging modalities on a pixel by pixel basis.

Proper citation: Elizabeth Neumann, Jamie Allen, Heath Patterson, Danielle Gutierrez, Jeff Spraggins 2020. Post-IMS Autofluorescence Microscopy. protocols.io https://dx.doi.org/10.17504/protocols.io.879hzr6 Copy   

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Authors: Kelley Knizner, Christopher Simmons
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community, GE Research
Summary: This method details sectioning of the FFPE HuBMAP specimens.Protocol adopted from Leigh Propper, Marda Jorgensen (12/13/2019). HuBMAP Tissue Sectioning for FFPE Specimens. https://dx.doi.org/10.17504/protocols.io.bahdib26

Proper citation: Kelley Knizner, Christopher Simmons 2020. HuBMAP | Sectioning of FFPE Specimens. protocols.io https://dx.doi.org/10.17504/protocols.io.bqqbmvsn Copy   

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Authors: Seth Currlin, Marda Jorgensen, Jerelyn Nick
Group: Optical Clearing of Tissue, Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: Tissue preparation for CLARITY includes fixation in 4% PFA, infusion with monomers in Hydrogel Solution, and clearing of lipids in Electrophoretic Tissue Clearing Solution.

Proper citation: Seth Currlin, Marda Jorgensen, Jerelyn Nick 2019. Tissue Preparation for CLARITY. protocols.io https://dx.doi.org/10.17504/protocols.io.8jihuke Copy   

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Authors: Jamie Allen, Elizabeth Neumann, Maya Brewer, Haichun Yang, Jeff Spraggins, Danielle Gutierrez
Group: VU Biomolecular Multimodal Imaging Center, Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: Scope:Prepare formalin fixed tissue with freezing fresh tissue protocol (dx.doi.org/10.17504/protocols.io.6wghfbw). This protocol provides the steps to prepare formalin fixed human kidney tissue and perform histology assessment for normalcy.Expected Outcome:Pathological assessment of kidney tissue for use in LC-MS/MS and imaging.

Proper citation: Jamie Allen, Elizabeth Neumann, Maya Brewer, Haichun Yang, Jeff Spraggins, Danielle Gutierrez 2020. Initial Rapid Pathology Assessment of Kidney Tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.9dph25n Copy   

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Authors: Seth Currlin, Marda Jorgensen, Jerelyn Nick
Group: Optical Clearing of Tissue, Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: The Zeiss Lightsheet 7 is used to quickly and efficiently scan large tissue volumes using laser light sheet illumination. The focused laser sheets and imaging plane are static while the tissue is moved throughout the selected volume for three dimensional image acquisition. This protocol will cover the procedures used for the day of light sheet image acquisition.Setting the acquisition volume: Large scanning volumes are composed of multiple z-stack being stitched back together based on overlapping edges of the scanning window. This requires setting an overall z-stack, which must include the entirety of tissue as covered by adjoining z-stacks. This becomes tricky when considering irregularly shaped tissues and their orientation relative to the various laser and imaging objectives.Dual-side light sheet alignment: To obtain a strong and clear image from large tissues it is helpful to illuminate the sample with both the left and right laser. Each laser sheet can be angled slightly within the focal plane to account for differences in the R.I. of the tissue and R.I. media (TDE). This is a manual tuning process and is not intended for major R.I. differences.Protocol Overview:- Refractive index matching of cleared tissue samples. - Mounting tissue onto sample holder.- Image acquisition in Zen 3.1 LS (black edition), including: - Setting the acquisition volume - Dual-sided light sheet alignment

Proper citation: Seth Currlin, Marda Jorgensen, Jerelyn Nick 2020. Light Sheet Fluorescence Microscopy Image Acquisition. protocols.io https://dx.doi.org/10.17504/protocols.io.bprsmm6e Copy   

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Authors: Elizabeth Neumann, Jamie Allen, Jeff Spraggins, Danielle Gutierrez
Group: VU Biomolecular Multimodal Imaging Center, Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: Scope: Annotate lipid species detected by MALDI IMS analysis.Expected Outcome:A list of lipid identifications with low mass errors (

Proper citation: Elizabeth Neumann, Jamie Allen, Jeff Spraggins, Danielle Gutierrez 2020. Lipid Annotation of MALDI IMS Datasets. protocols.io https://dx.doi.org/10.17504/protocols.io.864hzgw Copy   

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Authors: Michelle Daniel, Marda Jorgensen
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: This SOP describes the slide acquisition for spill-over correction of Imaging MassCytometry experiments. The protocol makes use of the compensation slide, which wasprepared previously following the SOP:“HuBMAP_Protocol_3_Compensation_Slide_Preparation.pdf” to measure eachconjugated antibody individually and to record all observed spill-over. The resultingcompensation matrix can be used to compensate data that has been recorded using thesame antibody conjugates.

Proper citation: Michelle Daniel, Marda Jorgensen 2020. Imaging Mass Cytometry Compensation Slide Acquisition. protocols.io https://dx.doi.org/10.17504/protocols.io.bf2jjqcn Copy   

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Authors: Leigh Propper, Marda Jorgensen
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: This method details the microtomy process for specimens that will be stained and analyzed using the Akoya CODEX® system.This process applies to paraffin embedded blocks containing tissue of no more than 1cm x 1cm in size.FFPE tissues for CODEX® analysis must be sectioned onto prepared poly-l-lysine coated coverslips.Cut and mounted tissue sections can be stored at 4°C for up to one(1 )month prior to staining.

Proper citation: Leigh Propper, Marda Jorgensen 2019. HuBMAP - Tissue Sectioning for CODEX Specimens. protocols.io https://dx.doi.org/10.17504/protocols.io.basdiea6 Copy   

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Authors: yang1832 , Julia Laskin
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community, NanoDESI MSI Julia Laskin group Purdue
Summary: Our goal is to map the biological tissues with Nano-DESI Mass Spectrometry Imaging and build an atlas of human kidney.Scope: Provide an overview of the sample preparation steps used by the Vanderbilt Tissue Mapping Center and Nano-DESI MSI workflow used by Julia Laskin's group as part of the Human Biomolecular Atlas Program (HuBMAP, NIH Common Fund).

Proper citation: yang1832 , Julia Laskin 2020. Nano-DESI Mass Spectrometry Imaging kidney characterization pipeline for tissues collected by Vanderbilt University. protocols.io https://dx.doi.org/10.17504/protocols.io.bmwik7ce Copy   

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Authors: John Hickey
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: See our detailed protocol published with the following title: CODEX multiplexed tissue imaging with DNA-conjugated antibodies.

Proper citation: John Hickey 2021. Sample prep - Stanford TMC. protocols.io https://dx.doi.org/10.17504/protocols.io.be9zjh76 Copy   

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Authors: Jamie Allen, Maya Brewer, Elizabeth Neumann, Danielle Gutierrez, Mark deCaestecker, Jeff Spraggins
Group: VU Biomolecular Multimodal Imaging Center, Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: Scope:To describe the procedure for freezing fresh tissue procured from the Cooperative Human Tissue Network.Expected Outcome:Frozen tissue should have minimal structural damage from freezing.

Proper citation: Jamie Allen, Maya Brewer, Elizabeth Neumann, Danielle Gutierrez, Mark deCaestecker, Jeff Spraggins 2019. Freezing Fresh Tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.6wghfbw Copy   

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Authors: Marda Jorgensen
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: This document outlines the required criteria for inclusion for donors of thymus, lymph nodes and spleen in the HuBMAP project.

Proper citation: Marda Jorgensen 2020. Donor Acceptance Criteria for TMC Florida/Zurich HuBMAP Inclusion. protocols.io https://dx.doi.org/10.17504/protocols.io.bipykdpw Copy   

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Authors: Marda Jorgensen
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: This SOP describes the methods used to ship paraffin-embedded blocks to the University of Zurich.

Proper citation: Marda Jorgensen 2020. Shipping Paraffin Blocks to the Bodenmiller Lab for IMC Analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.bf5njq5e Copy   

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Authors: John Hickey
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: See our detailed protocol published with the following title: CODEX multiplexed tissue imaging with DNA-conjugated antibodies.

Proper citation: John Hickey 2021. Reagent prep - Stanford TMC. protocols.io https://dx.doi.org/10.17504/protocols.io.be92jh8e Copy   

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