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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: 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: 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: 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: 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
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 dx.doi.org/10.17504/protocols.io.bf2gjqbw Copy
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 dx.doi.org/10.17504/protocols.io.bprsmm6e Copy
Authors: Franchesca Farris, Marda Jorgensen
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: UF H&E Staining
Proper citation: Franchesca Farris, Marda Jorgensen 2019. UF H&E Staining. protocols.io dx.doi.org/10.17504/protocols.io.8jthunn Copy
Authors: Marda Jorgensen, Franchesca Farris
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: FFPE tissues for CODEX® analysis must adhere directly onto Poly-lysine-coated coverslips. Preparation and storage of tissue slices are critical for sample integrity.
Proper citation: Marda Jorgensen, Franchesca Farris 2019. FFPE Tissue Sectioning for CODEX. protocols.io dx.doi.org/10.17504/protocols.io.8mfhu3n Copy
Authors: Franchesca Farris, Marda Jorgensen
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: This is the appendix for the Thymus SOP.
Proper citation: Franchesca Farris, Marda Jorgensen 2020. SOP Appendix for Thymus . protocols.io dx.doi.org/10.17504/protocols.io.bcwgixbw Copy
Authors: Leigh Propper, Franchesca Farris, Marda Jorgensen
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: Poly-Lysine Coverslip Preparation This section describes the process of creating Poly-lysine-coated coverslips that are used for the tissue sections in the CODEX® experiment workflow.
Proper citation: Leigh Propper, Franchesca Farris, Marda Jorgensen 2019. CODEX - Poly-l-Lysine Cover-Slip Preparation. protocols.io dx.doi.org/10.17504/protocols.io.baeribd6 Copy
Authors: Leigh Propper, Franchesca Farris, Marda Jorgensen
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: Poly-Lysine Coverslip Preparation This protocol describes the process of creating Poly-lysine-coated coverslips that are required for mounting tissue sections for the CODEX® experiment workflow.
Proper citation: Leigh Propper, Franchesca Farris, Marda Jorgensen 2020. CODEX - Poly-l-Lysine Cover-Slip Preparation. protocols.io dx.doi.org/10.17504/protocols.io.baxyifpw 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.bf3kjqkw 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 OCT embedding of HuBMAP frozen fixed specimens.
Proper citation: Marda Jorgensen, Jerelyn Nick 2020. HuBMAP: Embedding Fixed Frozen OCT Samples. protocols.io dx.doi.org/10.17504/protocols.io.basniede Copy
Authors: Franchesca Farris, Marda Jorgensen
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: Poly-Lysine Coverslip Preparation This section describes the process of creating Poly-lysine-coated coverslips that are used for the tissue slices in the CODEX® experiment workflow.
Proper citation: Franchesca Farris, Marda Jorgensen 2019. Poly-Lysine Coverslip Preparation. protocols.io dx.doi.org/10.17504/protocols.io.8kthuwn 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 dx.doi.org/10.17504/protocols.io.bbgnijve Copy
Authors: Leigh Propper, Marda Jorgensen
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: Microtomy Tracking Sheet for CODEX SpecimensThe process of sectioning CODEX specimens presents tediously at the tissue block trimming and microtomy stage.As you begin to cut into a new or already used tissue block, you must keep track of how many microns of tissue are removed from the section during trimming, and also during microtomy.Attached is a generic document that is used to keep record of what tissue block is being cut. All identification factors, trimming data, what tissue sections were used and their purpose, as well as storage information. Formal logging of how far into the tissue block you have traveled as well as how many sections were used is an extremely important and necessary function in this process.
Proper citation: Leigh Propper, Marda Jorgensen 2019. CODEX Microtomy Tracking Sheet. protocols.io dx.doi.org/10.17504/protocols.io.baefibbn Copy
Authors: Marda Jorgensen, Michelle Daniel
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: FFPE block are cut into thin sections.
Proper citation: Marda Jorgensen, Michelle Daniel 2020. Cutting FFPE Sections for Imaging Mass Cytometry. protocols.io dx.doi.org/10.17504/protocols.io.bfz4jp8w Copy
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