Apr 13, 2020
Version 1
Optical Image Collection V.1
- Guanshi Zhang1,
- Dusan Velickovic2,
- Annapurna Pamreddy1,
- Jessica Lukowski2,
- Theodore Alexandrov3,
- Christopher R Anderton2,
- Kumar Sharma1
- 1University of Texas Health San Antonio;
- 2Pacific Northwest National Laboratory;
- 3European Molecular Biology Laboratory
- KPMP
- Metabolomics Protocols & Workflows
Protocol Citation: Guanshi Zhang, Dusan Velickovic, Annapurna Pamreddy, Jessica Lukowski, Theodore Alexandrov, Christopher R Anderton, Kumar Sharma 2020. Optical Image Collection. protocols.io https://dx.doi.org/10.17504/protocols.io.beumjeu6
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 08, 2020
Last Modified: April 23, 2020
Protocol Integer ID: 35437
Abstract
Mass spectrometry imaging is an exciting technology, which enables simultaneous analysis of multiple molecular components directly from single cells, tissues, and organs. In combination with histological methods, this technique provides information about the spatial distribution of molecules in various biological tissues. Particularly, MALDI-MS imaging increases the coverage of metabolites by using different matrices and ionization modes. In coordination with in situ analysis of proteins, transcripts and epigenetic marks, the complementary spatial information on metabolites will establish metabolic pathways that are dominant and characteristic of disease states. We have recently developed and optimized a spatial metabolomics approach to image small molecules in human kidneys and biopsy sized material. With our combined expertise at UTHSA, PNNL and EMBL and recent advances, we have established methods for identifying metabolites in human kidneys, employed ultra-high mass resolution MS imaging for tissue analysis, and developed a bioinformatics resource (METASPACE) to annotate metabolites for anatomical localization and 3-D reconstruction. Our integrated technology can easily connect with other TIS sites to provide biochemical readouts of genes/proteins in specific tissue and cellular compartments.
Guidelines
Overview
1. AF and bright field images are captured on a confocal microscope before the matrix application.
2. H&E and PAS staining
a) H&E and PAS staining is performed on both the serial section glass slide mounted samples and MALDI analyzed samples.
b) MALDI analyzed samples are washed with 70% EtOH, 2 min, 3x to remove matrix
c) Washed slides are dried in vacuum dessicator for 20 minutes prior to staining
3. Defrosted sections on glass slides are used as is.
Materials
MATERIALS
100% Ethanol
NovaUltra H&E Stain KitIHC WorldCatalog #IW-3100
NovaUltra PAS Stain KitIHC WorldCatalog #IW-3019
Eosin Solution
Mayers hematoxylin solution
Mastertech Bluing SolutionAmerican Master Tech ScientificCatalog #HXB00288E
Mastertech Differentiating SolutionCatalog #HXD00188E
BBC Biochemical Optic Mount I XyleneScientific Supply & EquipmentCatalog #BBC 7722
95% EthanolThermo Scientific
10% Formalin
0.5% Periodic Acid Solution (0.5g periodic acid in 100mL DI water)
37% Formalin
Coverslips
Chemical Permount Mounting MediumFisher ScientificCatalog #SP15-100
Aperio ScanScope
Leica TCS SP8 Confocal Microscope
STEP MATERIALS
Mayers hematoxylin solution
Mastertech Differentiating SolutionCatalog #HXD00188E
Mastertech Bluing SolutionAmerican Master Tech ScientificCatalog #HXB00288E
Eosin Solution
75% Ethanol
95% EthanolThermo Scientific
100% Ethanol
Xylene
Coverslips
Chemical Permount Mounting MediumFisher ScientificCatalog #SP15-100
0.5% Periodic Acid Solution (0.5g periodic acid in 100mL DI water)
Schiff’s reagent Merck MilliporeSigma (Sigma-Aldrich)Catalog #3952016-500ML
Mayers hematoxylin solution
95% EthanolThermo Scientific
100% Ethanol
Xylene
Coverslips
Chemical Permount Mounting MediumFisher ScientificCatalog #SP15-100
75% Ethanol
Protocol materials
Mastertech Differentiating SolutionCatalog #HXD00188E
37% Formalin
Mastertech Differentiating SolutionCatalog #HXD00188E
Eosin Solution
Mastertech Bluing SolutionAmerican Master Tech ScientificCatalog #HXB00288E
10% Formalin
Coverslips
Aperio ScanScope
Chemical Permount Mounting MediumFisher ScientificCatalog #SP15-100
Coverslips
0.5% Periodic Acid Solution (0.5g periodic acid in 100mL DI water)
95% EthanolThermo Scientific
Mayers hematoxylin solution
95% EthanolThermo Scientific
Chemical Permount Mounting MediumFisher ScientificCatalog #SP15-100
NovaUltra PAS Stain KitIHC WorldCatalog #IW-3019
Xylene
Schiff’s reagent Merck MilliporeSigma (Sigma-Aldrich)Catalog #3952016-500ML
NovaUltra H&E Stain KitIHC WorldCatalog #IW-3100
75% Ethanol
Mayers hematoxylin solution
Xylene
Chemical Permount Mounting MediumFisher ScientificCatalog #SP15-100
Mayers hematoxylin solution
Mastertech Bluing SolutionAmerican Master Tech ScientificCatalog #HXB00288E
100% Ethanol
Coverslips
Eosin Solution
100% Ethanol
75% Ethanol
BBC Biochemical Optic Mount I XyleneScientific Supply & EquipmentCatalog #BBC 7722
100% Ethanol
Leica TCS SP8 Confocal Microscope
95% EthanolThermo Scientific
0.5% Periodic Acid Solution (0.5g periodic acid in 100mL DI water)
Mayers hematoxylin solution
Mastertech Differentiating SolutionCatalog #HXD00188E
Mastertech Bluing SolutionAmerican Master Tech ScientificCatalog #HXB00288E
Eosin Solution
75% Ethanol
95% EthanolThermo Scientific
100% Ethanol
Xylene
Coverslips
Chemical Permount Mounting MediumFisher ScientificCatalog #SP15-100
0.5% Periodic Acid Solution (0.5g periodic acid in 100mL DI water)
Schiff’s reagent Merck MilliporeSigma (Sigma-Aldrich)Catalog #3952016-500ML
Mayers hematoxylin solution
Chemical Permount Mounting MediumFisher ScientificCatalog #SP15-100
75% Ethanol
95% EthanolThermo Scientific
100% Ethanol
Xylene
Coverslips
Autofluorescence (AF) and Bright Field Imaging
Autofluorescence (AF) and Bright Field Imaging
Before the matrix application, right after defrosting in desiccator, the AF image (pre-AF) is captured on a confocal microscope.
Note
UTHSA: 10Ă— objective on a Leica TSC SP8
PNNL: 20x objective on a Zeiss 710 LSM; 10x objective PALM MicroBeam (Zeiss inverse microscope Axiovert)
495 nm-720 nm, 404-488 nm and bright field channels are used for AF imaging with pinhole size 239 ÎĽm.
Matrix application and MALDI-MSI (duplicates for each sample at each spatial resolution).
AF image are uploaded to METASPACE and registered to corresponding MSI data.
H&E Staining
H&E Staining
Air dry slides (after storage at -80 °C ) for 00:03:00 to 00:05:00 under the hood.
Fix slides in ice cold acetone:methanol solution (1:1) for 00:10:00 to00:15:00 at -20 °C
Wash/rehydrate slides in distilled H2O for 00:10:00 to00:15:00
Stain slides with hematoxylin for 00:00:20 to00:00:30
Mayers hematoxylin solution
Wash slides with distilled H2O for 00:02:00
Rinse slides with differentiating solution by moving slides up and down 3-4 times within 00:00:05 . Then check the intensity of nuclear stianing under the microscope. If signal is too week, then go back to step 8 and do staining again for 00:00:05 to 00:00:10 , then rinse slides with distilled water again for 00:00:01 to 00:00:02 .
Mastertech Differentiating SolutionCatalog #HXD00188E
Wash slides with distilled H2O for 00:02:00
Rinse slides with Bluing solution by moving slides up and down for 00:00:05 to 00:00:10
Mastertech Bluing SolutionAmerican Master Tech ScientificCatalog #HXB00288E
Wash slides with distilled H2O for 00:02:00
Stain slides with Eosin for 00:00:15 to 00:00:20
Eosin Solution
Wash/dehydrate slides with 75% ethanol for 00:01:00 to 00:02:00
75% Ethanol
Dehydrate slides with 95% ethanol for 00:01:00 to 00:02:00
95% EthanolThermo Scientific
Dehydrate slides with 100% ethanol for 00:02:00 (twice)
100% Ethanol
Immerse slides in xylene for 00:02:00 (twice)
Xylene
Air dry slides under the hood for 00:03:00 to 00:05:00
Mount sections with Permount and cover with coverslips
Coverslips
Chemical Permount Mounting MediumFisher ScientificCatalog #SP15-100
Stained amples are imaged using an Aperio ScanScope XT
H&E images are uploaded to METASPACE and registered to the corresponding MSI data
Slides are then re-stained with PAS
PAS Staining
PAS Staining
Place the slides in 10% formalin in 95% alcohol for 00:10:00 .
Rinse in tap water four times.
Oxidize in 0.5% periodic acid solution for 00:05:00
0.5% Periodic Acid Solution (0.5g periodic acid in 100mL DI water)
Rinse in tap water four times.
Place in Schiff reagent for 00:15:00
Schiff’s reagent Merck MilliporeSigma (Sigma-Aldrich)Catalog #3952016-500ML
Note
Sections become a light pink color
Wash in lukewarm tap water for 00:05:00
Note
Sections immediatly become a dark pink color
Counterstain in Mayer's hematoxylin for 00:03:00
Mayers hematoxylin solution
Rinse in TBS solution and then rinse in deionized water four times.
Dehydrate by the successive ethanol and xylene steps 14 to 18 and coverslip using a synthetic mounting medium.
75% Ethanol
95% EthanolThermo Scientific
100% Ethanol
Xylene
Coverslips
Chemical Permount Mounting MediumFisher ScientificCatalog #SP15-100
PAS slides from same tissue section as the H&E slide is imaged using Aperio ScanScope XT and uploaded to METASPACE and registered to the corresponding data.