HuBMAP | GE/Vanderbilt MALDI IMS and Cell DIVE™ Modality Overview
Elizabeth Neumann, Nathan Heath Patterson, Jamie Allen, Jeff Spraggins, Liz McDonough, Christine Surrette, Soumya Ghose, Fiona Ginty
Abstract
This is an overview of all protocols currently in use for the GE/Vanderbilt University Cell DIVE collaboration for the Human BioMolecular Atlas Program (HuBMAP). It includes links to each of the individual protocols that make up this project workflow.
Steps
MALDI IMS
Collection of post-surgical tissue.
Collection and Post-Surgical Excision of Human Kidney Tissue through the Cooperative Human Tissue Network
Stabilize and freeze tissues.
Freezing Fresh Tissue
Cryosection tissues into micrometer thick sections, alternating between thaw mounting onto indium tin-oxide and positively charged glass slides (proceed to step 4), or collecting several tissue sections within an microcentrifuge tube for proteomics analysis.
Cryostat Sectioning of Tissues for 3D Multimodal Molecular Imaging
Perform autofluorescence microscopy on all tissue sections
Autofluorescence Microscopy Data Acquisition
Perform Matrix Application
Automatic Deposition of CHCA Matrix for MALDI Analysis of Lipids
Perform high resolution IMS analysis of matrix coated tissue sections.High Resolution Imaging Mass Spectrometry Analysis using Bruker Daltonics Platforms
Obtain autofluorescence microscopy images of tissues after IMS analysis
Post-IMS Autofluorescence Microscopy
Preparing Sample for MxIF
Perform Matrix Removal & Tissue Fixation
Fixation Protocol for Fresh Frozen Tissue Samples (post-MALDI)
Cell DIVE
Characterize antibodies (primary/secondary, direct conjugates, and zenon labelled antibodies) and determine any antigen effects from the Cell DIVE dye inactivation process.
Cell DIVE™ Platform | Antibody Characterization for Multiplexing
Cell DIVE™ Platform | Antibody Staining & Imaging
Prepare direct conjugates for study.
Cell DIVE™ Platform | Antibody Purification Chemistry
Cell DIVE™ Platform | Ab Conjugation: Initial Conjugation & Scale up Conjugation
Perform Cell DIVE™ multiplexed data acquisition on the final cohort.
