Apr 29, 2020
Version 4
VU Biomolecular Multimodal Imaging Center (BIOMIC) kidney characterization pipeline for tissues collected through the Cooperative Human Tissue Network (CHTN) V.4
- Elizabeth Neumann1,
- Jamie Allen1,
- Maya Brewer1,
- David Anderson1,
- Mark De Caestecker2,
- Danielle Gutierrez1,
- Jeff Spraggins1
- 1Vanderbilt University;
- 2Division of Nephrology, Vanderbilt University Medical Center
- VU Biomolecular Multimodal Imaging Center / Spraggins Research Group
- Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Protocol Citation: Elizabeth Neumann, Jamie Allen, Maya Brewer, David Anderson, Mark De Caestecker, Danielle Gutierrez, Jeff Spraggins 2020. VU Biomolecular Multimodal Imaging Center (BIOMIC) kidney characterization pipeline for tissues collected through the Cooperative Human Tissue Network (CHTN). protocols.io https://dx.doi.org/10.17504/protocols.io.bfskjncw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 29, 2020
Last Modified: October 18, 2023
Protocol Integer ID: 36396
Keywords: HuBMAP, BIOMIC, Vanderbilt, Multimodal Imaging, IMS, MxIF, Proteomics, Image Registration
Abstract
We aim to develop high resolution, chemically informative imaginig methodologies for building an atlas of human organs, such as the kidney.
Scope:
Provide an overview of the methods used by the Vanderbilt Tissue Mapping Center as part of the Human Biomolecular Atlas Program (HuBMAP, NIH Common Fund) and contextualize individual protocols within our larger workflow.
Collection of post-surgical tissue.
Collection: dx.doi.org/10.17504/protocols.io.7gehjte
Intial Rapid Pathology Assessment of Kidney Tissue
Staining: dx.doi.org/10.17504/protocols.io.4qngvve
Assessment: dx.doi.org/10.17504/protocols.io.9dph25n
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.
Cryosectioning: dx.doi.org/10.17504/protocols.io.7ethjen
Perform LC based Proteomics on adjacent tissue sections and/or continue to Step 6.
Sample Preparation: dx.doi.org/10.17504/protocols.io.67nhhme
Data Acquisition: dx.doi.org/10.17504/protocols.io.bfsjjncn
Data Analysis: dx.doi.org/10.17504/protocols.io.bfshjnb6
Perform autofluorescence microscopy on all tissue sections before IMS (step 5) or MxIF analysis (step 9)
Autofluorescence: dx.doi.org/10.17504/protocols.io.7e3hjgn
Coat tissue sections with MALDI matrix for IMS analysis.
Matrix Application: dx.doi.org/10.17504/protocols.io.4srgwd6
Perform high resolution IMS analysis of matrix coated tissue sections.
Perform fluorescence microscopy to visualize laser ablation spots.
Post IMS AF: dx.doi.org/10.17504/protocols.io.879hzr6
Annotation of Lipids from IMS Data
Cal & Annotate: dx.doi.org/10.17504/protocols.io.864hzgw
Remove MALDI matrix and perform PAS staining.
PAS Staining: dx.doi.org/10.17504/protocols.io.4qngvve
Alternatively, MxIF can be peformed after step 4.
Antibody labeling: dx.doi.org/10.17504/protocols.io.667hhhn
Registration of autofluorescence images from both IMS and MxIF sections allow for the direct correlation of the two orthogonal approaches.
Registration: https://dx.doi.org/10.17504/protocols.io.bed2ja8e
Publication: https://doi.org/10.1021/acs.analchem.8b02884
MALDI imaging mass spectrometry data processing.
Data Processing: https://dx.doi.org/10.17504/protocols.io.bed3ja8n
RNA Assessment from tissue.
RNA extraction: dx.doi.org/10.17504/protocols.io.86nhzde