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Authors: Marda Jorgensen, Jerelyn Nick
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: The purpose of this Standard Operating Procedure (SOP) is to outline the procedures for processing and storing thymus tissue received for HuBMAP consortium assay and analysis.
Proper citation: Marda Jorgensen, Jerelyn Nick 2020. Case Processing SOP for Thymus . protocols.io dx.doi.org/10.17504/protocols.io.bbgmiju6 Copy
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 dx.doi.org/10.17504/protocols.io.bf2jjqcn Copy
Authors: Jerelyn Nick, Marda Jorgensen
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: A CODEX® Reporter is a fluorescent dye conjugated to an oligonucleotide that is complementary to a specific oligonucleotide barcoded antibody. This protocol describes how to prepare 96-well plates of Reporter Mix for use in a CODEX® run. Each well contains Reporters are grouped together in mixtures of three per cycle plus the nuclear stain. During each cycle the Codex instrument dispenses one well on the tissue sample and images the 3 markers and the nuclear stain. At the end of the cycle the Reporters are removed from the tissue by a gentle wash.
Proper citation: Jerelyn Nick, Marda Jorgensen 2020. CODEX Preparation of Reporter 96-Well Plates. protocols.io dx.doi.org/10.17504/protocols.io.bc2riyd6 Copy
Authors: Marda Jorgensen, Maigan Brusko
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Proper citation: Marda Jorgensen, Maigan Brusko 2020. HuBMAP UF TMC - 10x Genomics scRNAseq Modality Overview . protocols.io dx.doi.org/10.17504/protocols.io.be79jhr6 Copy
Authors: Marda Jorgensen, Jerelyn Nick
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: The purpose of this Standard Operating Procedure (SOP) is to outline procedures for the paraffin embedding of HuBMAP specimens.
Proper citation: Marda Jorgensen, Jerelyn Nick 2020. HuBMAP: Paraffin Embedding Tissue Samples . protocols.io dx.doi.org/10.17504/protocols.io.bam9ic96 Copy
Authors: Michelle Daniel, Marda Jorgensen
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: This SOP describes the image acquisition with an Imaging Mass Cytometer (IMC orHyperion).Prior to this protocol the IMC needs to be fully tuned to specifications (see“HuBMAP_Protocol_4_IMC_Tuning.pdf”). Additionally, every batch of samples stainedwith the same antibody-mix requires the acquisition of a compensation slide for spill overcorrection (see “HuBMAP_Protocol_3_Compensation_Slide_Preparation.pdf” &“HuBMAP_Protocol_6_IMC_Compensation_Slide_Acquisition.pdf”).
Proper citation: Michelle Daniel, Marda Jorgensen 2020. Imaging Mass Cytometry Data Acquisition. protocols.io dx.doi.org/10.17504/protocols.io.bf2ijqce Copy
Authors: Marda Jorgensen
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: This is the appendix for lymph nodes.
Proper citation: Marda Jorgensen 2020. SOP Appendix for Lymph Node. protocols.io dx.doi.org/10.17504/protocols.io.bcwpixdn Copy
Authors: Marda Jorgensen
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: The purpose of this Standard Operating Procedure is to outline procedures for processing and storing spleen received for HuBMAP consortium assay and analysis.
Proper citation: Marda Jorgensen 2020. Case Processing SOP (Spleen). protocols.io dx.doi.org/10.17504/protocols.io.bc3kiykw Copy
Authors: Marda Jorgensen, Jerelyn Nick, Franchesca Farris, Jesus Penaloza
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: This protocol is an overview of all of the protocols currently in use for the CODEX modality at HubMAP Tissue Mapping Center,TMC-Florida/Zurich. It includes protocols.io links to each of the individual protocols that make up this project workflow, from processing tissue and imaging the samples, to uploading the final data to the HuBMAP HIVE.
Proper citation: Marda Jorgensen, Jerelyn Nick, Franchesca Farris, Jesus Penaloza 2020. HuBMAP TMC-Florida/Zurich CODEX Modality Overview. protocols.io dx.doi.org/10.17504/protocols.io.be9pjh5n Copy
Authors: Seth Currlin, Marda Jorgensen
Group: Optical Clearing of Tissue, Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: In order to track tissue position and orientation each sample is photographed when received for lightsheet microscopy (Figure 1, A). Large samples (>1 cm3), such as the spleen in Figure 1, are cut into 2 mm sections (Figure 1, B) to facilitate tissue clearing, staining, and three-dimensional imaging. These tissues are batch processed and withdrawn from the lipid clearing pipeline as needed (Figure 1, C); at this time a novel identifier is assigned to the sample.
Proper citation: Seth Currlin, Marda Jorgensen 2020. Lightsheet Tissue Intake - Photodocumentation and Tracking. protocols.io dx.doi.org/10.17504/protocols.io.bef6jbre Copy
Authors: Marda Jorgensen, Franchesca Farris
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: Hematoxylin and Eosin stains are the prefered method for histopathologic assessment of tissue sections.Hematoxylin is used to illustrate nuclear detail in cells. The resulting intensity and shade of the dye in the cells is primarily reliant on the length of time the sample spends in hematoxylin. The cytoplasmic component of the section is identified by Eosin, which is the most commonly used counterstain in histology. It's varying shades of pink and red provide an exceptional contrast between the nuclear and cytoplasmic areas of interest.
Proper citation: Marda Jorgensen, Franchesca Farris 2020. UF H&E Staining. protocols.io dx.doi.org/10.17504/protocols.io.beamjac6 Copy
Authors: Jerelyn Nick, Franchesca Farris, Marda Jorgensen
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: Standard IHC staining, such as this DAB protocol, is included in the validation of new antibodies at the University of Florida Tissue Mapping Center, TMC-Florida/Zurich. This procedure includes all of the reagents necessary to perform a DAB based immunohistochemistry reaction. DAB Substrate (3,3’-diaminobenzidine) produces a brown reaction product in the presence of peroxidase (HRP) enzyme.
Proper citation: Jerelyn Nick, Franchesca Farris, Marda Jorgensen 2020. DAB Staining of FFPE Slides. protocols.io dx.doi.org/10.17504/protocols.io.bdiei4be Copy
Authors: Leigh Propper, Marda Jorgensen
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: This method details our microtomy (sectioning) process for reasearch specimens involved with HuBMAP.This process follows the microtomy process that follows after receiving, processing, and embedding of research specimens into formalin fixed, paraffin embedded tissue blocks.
Proper citation: Leigh Propper, Marda Jorgensen 2019. HuBMAP Tissue Sectioning for FFPE Specimens. protocols.io dx.doi.org/10.17504/protocols.io.bahdib26 Copy
Authors: Marda Jorgensen, Franchesca Farris, Jerelyn Nick
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: The purpose of this Standard Operating Procedure (SOP) is to outline procedures for the automated processing of formalin fixed HuBMAP specimens for paraffin embedding.
Proper citation: Marda Jorgensen, Franchesca Farris, Jerelyn Nick 2020. HuBMAP - Tissue Processing with Paraffin Infiltration. protocols.io dx.doi.org/10.17504/protocols.io.8nzhvf6 Copy
Authors: Marda Jorgensen
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: The purpose of this Standard Operating Procedure is to outline procedures for processing and storing spleen received for HuBMAP consortium assay and analysis.
Proper citation: Marda Jorgensen 2020. Case Processing SOP (Spleen). protocols.io dx.doi.org/10.17504/protocols.io.bbcuiiww Copy
Authors: Marda Jorgensen, Jerelyn Nick
Summary: This protocol describes the methods for validating antibodies after custom conjugation with Akoya CODEX barcodes in FFPE tissues on coverslips. Included are the stepwise protocols for pre-staining, deparaffinization, antigen retrival, antibody staining, post-fixation, manual addition of CODEX Reporters and tissue mounting on microscope slides. The recommended single stain experimental design includes 3 tissues sections from the same block stained with the CODEX-tagged antibody alone (1), co-stained with a positive control(2), and co-satined with a negative control(3). ABCD1 Tissue 1Tissue 2Tissue 32 CODEX-Stain Only CODEX-stain
with co-stain (Positive Control) CODEX-stain
with counter-stain (Negative Control)3DAPI ChannelNuclear StainNuclear StainNuclear Stain42nd
Fluorescent ChannelCODEX-tagged AnitbodyCODEX-tagged AnitbodyCODEX-tagged Anitbody53rd or 4th
Fluorescent ChannelNone Control
antibody targeting a different antigen expressed by the same phenotype or
cell populationControl
antibody targeting an antigen expressed by a different phenotype or cell
population.The enitre process requires approximately 5.5 hours, including a 2 hour incubation. Except as noted, reagents and consumables should be prepared before begining the protocol.
Proper citation: Marda Jorgensen, Jerelyn Nick 2020. HuBMAP TMC-UF Validation of Custom conjugated Antibodies for CODEX. protocols.io dx.doi.org/10.17504/protocols.io.bkpzkvp6 Copy
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 dx.doi.org/10.17504/protocols.io.8kvhuw6 Copy
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 dx.doi.org/10.17504/protocols.io.bfz8jp9w Copy
Authors: Leigh Propper, Marda Jorgensen
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: This method details our microtomy (sectioning) process for reasearch specimens involved with HuBMAP.This process follows the microtomy process that follows after receiving, processing, and embedding of research specimens into formalin fixed, paraffin embedded tissue blocks.
Proper citation: Leigh Propper, Marda Jorgensen 2019. HuBMAP Tissue Sectioning for FFPE Specimens. protocols.io dx.doi.org/10.17504/protocols.io.93qh8mw Copy
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 dx.doi.org/10.17504/protocols.io.bipykdpw Copy
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