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Authors: Jamie Allen, Elizabeth Neumann, Jeff Spraggins
Group: VU Biomolecular Multimodal Imaging Center, Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: Scope:To describe the procedure for spraying tissue sections with DAN for imaging lipids.Expected Outcome:Slides should be coated with DAN. Tissue sections should be imaged within 24 hours of matrix application.
Proper citation: Jamie Allen, Elizabeth Neumann, Jeff Spraggins 2019. Automatic Deposition of DAN Matrix using a TM Sprayer for MALDI Analysis of Lipids. protocols.io https://dx.doi.org/10.17504/protocols.io.4srgwd6 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 https://dx.doi.org/10.17504/protocols.io.bprtmm6n 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 https://dx.doi.org/10.17504/protocols.io.93qh8mw 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 https://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 https://dx.doi.org/10.17504/protocols.io.beamjac6 Copy
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.z3pf8mn 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 https://dx.doi.org/10.17504/protocols.io.bc2riyd6 Copy
Authors: Marda Jorgensen, Jerelyn Nick
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 lymph node tissue received for HuBMAP consortium assay and analysis.
Proper citation: Marda Jorgensen, Jerelyn Nick 2020. Case Processing SOP for Lymph Nodes . protocols.io https://dx.doi.org/10.17504/protocols.io.bbgnijve Copy
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