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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 dx.doi.org/10.17504/protocols.io.bpyxmpxn Copy
Authors: Seth Currlin, Marda Jorgensen, Jerelyn Nick
Group: Optical Clearing of Tissue, Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: This is a guide for immunostaining CLARITY-processed samples. dx.doi.org/10.17504/protocols.io.8jihukeThese steps are meant to be a guide for immunostaining large samples and should be optimized to suit your particular tissues and reagents. Large tissue volumes and dense tissue types will require longer incubation and wash times. The parameters suggested below are for a piece of human tissue (thymus, spleen, or lymph node) approximately 5 mm3 in size.A useful link: http://wiki.claritytechniques.org/index.php/Immunostaining
Proper citation: Seth Currlin, Marda Jorgensen, Jerelyn Nick 2020. Large Volume Immunostaining for Cleared Samples. protocols.io dx.doi.org/10.17504/protocols.io.bprtmm6n Copy
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 dx.doi.org/10.17504/protocols.io.8jihuke Copy
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 dx.doi.org/10.17504/protocols.io.bqqbmvsn Copy
Authors: Franchesca Farris
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: This is an appendix for spleen SOP.
Proper citation: Franchesca Farris 2020. Appendix for Spleen SOP. protocols.io dx.doi.org/10.17504/protocols.io.bbcwiixe Copy
Authors: Nina Dar, Long Cai
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: Once a tissue is received, it should be frozen using the following protocol:
Proper citation: Nina Dar, Long Cai 2019. Tissue Freezing for seqFISH. protocols.io dx.doi.org/10.17504/protocols.io.7mdhk26 Copy
Authors: Kelley Knizner, Christopher Simmons
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community, GE Research
Summary: This method details formalin fixation and paraffin embedding of the HuBMAP tissue specimens.Protocol adopted from Marda Jorgensen, Jerelyn Nick (02/24/2020). HuBMAP: Paraffin Embedding Tissue Samples . https://dx.doi.org/10.17504/protocols.io.bam9ic96
Proper citation: Kelley Knizner, Christopher Simmons 2020. HuBMAP | Formalin Fixation and Paraffin Embedding of Tissue Samples . protocols.io dx.doi.org/10.17504/protocols.io.bqp6mvre Copy
Authors: Michelle Daniel, Marda Jorgensen
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Proper citation: Michelle Daniel, Marda Jorgensen 2020. Imaging Mass Cytometry Modality Overview . protocols.io dx.doi.org/10.17504/protocols.io.bgatjsen Copy
Authors: Franchesca Farris, Marda Jorgensen, Jerelyn Nick, Jesus Peñaloza
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: Multiplex imaging of lymph node, thymus and spleen is accomplished using the Akoya Biosciences CODEX system at the HubMAP Tissue Mapping Center at the University of Florida, TMC-UF.This protocol is an overview of the CODEX raw data acquisition process guided by the CODEX Instrument Manager (CIM) , which is part of the CODEX Software Suite. The CODEX Instrument Manager is necessary to perform CODEX experiments. It controls the fluidics of the CODEX instrument the integration and synchronization with the Keyence microscope, image data formatting and facilitates transfer of data to the analysis computer.For more information, consult the Akoya Biosciences CODEX User Manual - A.0.
Proper citation: Franchesca Farris, Marda Jorgensen, Jerelyn Nick, Jesus Peñaloza 2020. CODEX Acquisition Protocol. protocols.io dx.doi.org/10.17504/protocols.io.bdifi4bn Copy
Authors: Gloria Pryhuber
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community, LungMap2 Consortium, URMC Pryhuber Lab
Summary: Purpose and Scope of the ProcedureStandardize process for receiving lung donations Scope: Coordination of screening, acceptance and receipt of tissue for research programsPrinciples Lung donations rejected for transplantation can be used to understand human lung structure and development, ultimately to improve care of patients with lung diseaseRapid, standardized and safe processing of human tissue for the HTC requires coordination of a team of staff, materials and attention to protocolsRapid processing is required to maintain the tissues in a state as close to normal as possibleUltra-high resolution CT Scan in an inflated state will provide a high-level comparative assessment of human lung structure across developmental ages (See Protocol 603)Principles Lung donations rejected for transplantation can be used to understand human lung structure and development, ultimately to improve care of patients with lung diseaseRapid, standardized and safe processing of human tissue for the HTC requires coordination of a team of staff, materials and attention to protocolsRapid processing is required to maintain the tissues in a state as close to normal as possibleUltra-high resolution CT Scan in an inflated state will provide a high-level comparative assessment of human lung structure across developmental ages (See Protocol 603)
Proper citation: Gloria Pryhuber 2020. 602.2 Donor Acceptance Criteria for URMC HTC HuBMAP and LungMAP Inclusion. protocols.io dx.doi.org/10.17504/protocols.io.bjuxknxn Copy
Authors: Maya Brewer, Jamie Allen, Elizabeth Neumann, Agnes Fogo, Raymond Harris, Danielle Gutierrez, Mark De Caestecker, Jeff Spraggins
Group: VU Biomolecular Multimodal Imaging Center, Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: Scope:Obtain kidney tissue and metadata about tissue location within the whole kidney for storage and analysis.Expected Outcome:A portion of kidney tissue and a series of images that provide information about the original location of the smaller tissue.
Proper citation: Maya Brewer, Jamie Allen, Elizabeth Neumann, Agnes Fogo, Raymond Harris, Danielle Gutierrez, Mark De Caestecker, Jeff Spraggins 2019. Collection and Post-Surgical Excision of Human Kidney Tissue through the Cooperative Human Tissue Network. protocols.io dx.doi.org/10.17504/protocols.io.7gehjte Copy
Authors: Anup Sood, Eric Williams, Liz McDonough
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community, GE Research
Summary: The purpose of the protocol is to purify antibodies that will be conjugated to Cy dyes as per the Cell DIVE™ technology. Affinity chromatography will be used to remove impurities from the vendor antibody to enable conjugation.
Proper citation: Anup Sood, Eric Williams, Liz McDonough 2021. Cell DIVE™ Platform | Antibody Purification Chemistry. protocols.io dx.doi.org/10.17504/protocols.io.bpx9mpr6 Copy
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. Imaging - Stanford TMC. protocols.io dx.doi.org/10.17504/protocols.io.be8qjhvw Copy
Authors: Franchesca Farris, Maigan Brusko
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: This protocol is for the generation of 3' (chemistry version 3) cDNA libraries from individual cells via droplet generation utilizing the 10x Genomics Chromium Controller and associated reagents. Paired end libraries are then sequenced on Illumina instruments.
Proper citation: Franchesca Farris, Maigan Brusko 2020. HuBMAP UF TMC - Single Cell RNA Seq (10x Genomics) Library Preparation and Sequencing . protocols.io dx.doi.org/10.17504/protocols.io.bffvjjn6 Copy
Authors: Maya Brewer, Yuantee Zhu, Elizabeth Neumann, Danielle Gutierrez, Jeff Spraggins, Mark De Caestecker
Group: VU Biomolecular Multimodal Imaging Center, Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: Scope:To describe the procedure for multiple cycles of immunofluorescence on human kidney tissue embedded in carboxymethylcellulose.Expected Outcome:Kidney tissue sections that have been tagged with antibodies for imaging microscopy.
Proper citation: Maya Brewer, Yuantee Zhu, Elizabeth Neumann, Danielle Gutierrez, Jeff Spraggins, Mark De Caestecker 2019. Multiplex Immunofluorescence on Fresh Frozen Tissue. protocols.io dx.doi.org/10.17504/protocols.io.665hhg6 Copy
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 dx.doi.org/10.17504/protocols.io.864hzgw Copy
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 dx.doi.org/10.17504/protocols.io.basdiea6 Copy
Authors: Marda Jorgensen, Jerelyn Nick
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: This protocol describes the method for antibody staining of FFPE tissues on coverslips using CODEX Barcoded Antibodies. Included are the stepwise protocols for pre-staining, deparaffinization, antigen retrival, antibody staining and post-fixation. The enitre process requires approximately 6.5 hours, including a 3 hour incubation. Except as noted, reagents and consumables should be prepared before being the protocol. Stained tissues can be stored in CODEX Storage Buffer at 4º C for up to 2 weeks for use in CODEX multiplex imaging.
Proper citation: Marda Jorgensen, Jerelyn Nick 2020. CODEX Antibody Staining Protocol for FFPE tissues. protocols.io dx.doi.org/10.17504/protocols.io.bbsdina6 Copy
Authors: Gloria Pryhuber, Heidie Huyck, Lisa Rogers, Cory Poole
Group: Human Cell Atlas Method Development Community, Human BioMolecular Atlas Program (HuBMAP) Method Development Community, LungMap2 Consortium, URMC Pryhuber Lab
Summary: Purpose and Scope of the Procedure- Rapid blocking, embedding and freezing of human lung tissue in no freezing media, 100% OCT or 5% CMC - Rapid freezing of non-lung tissue in no freezing media, 100% OCT or 5% CMC
Proper citation: Gloria Pryhuber, Heidie Huyck, Lisa Rogers, Cory Poole 2020. 615.1 URMC HTC Non-Inflated Fresh-Frozen Embedded Lung and Associated Tissue. protocols.io dx.doi.org/10.17504/protocols.io.bjtnknme Copy
Authors: Seth Currlin, Marda Jorgensen, Jerelyn Nick
Group: Optical Clearing of Tissue, Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: This document covers the CLARITY protocol for volumetric tissue clearing. This method can be applied to many tissue types and is easily modified. The link below is very useful and should be reviewed before beginning any CLARITY process. http://wiki.claritytechniques.org/index.php/CLARITY_Technique
Proper citation: Seth Currlin, Marda Jorgensen, Jerelyn Nick 2020. Tissue Clearing Using CLARITY Method. protocols.io dx.doi.org/10.17504/protocols.io.bppimmke Copy
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