TITLE: Optical Image Collection
AUTHORS: Guanshi Zhang, Dusan Velickovic, Annapurna Pamreddy, Jessica Lukowski, Theodore Alexandrov, Chris Anderton, Kumar Sharma

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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.</div></div>

[STEPS]
?. [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.
UTHSA: 10× objective on a Leica TSC SP8PNNL: 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 μm.
?. [Autofluorescence (AF) and Bright Field Imaging]
Matrix application and MALDI-MSI (duplicates for each sample at each spatial resolution).
?. [Autofluorescence (AF) and Bright Field Imaging]
AF image are uploaded to METASPACE and registered to corresponding MSI data.
?. [H&E Staining]
Air dry slides (after storage at  ) for  to  under the hood.
-80 °C
?. [H&E Staining]
Fix slides in ice cold acetone:methanol solution (1:1) for  to at
-20 °C
?. [H&E Staining]
Wash/rehydrate slides in distilled H2O for  to
?. [H&E Staining]
Stain slides with hematoxylin for  to
?. [H&E Staining]
Wash slides with distilled H2O for
?. [H&E Staining]
Rinse slides with differentiating solution by moving slides up and down 3-4 times within  . 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  to  , then rinse slides with distilled water again for  to  .
?. [H&E Staining]
Wash slides with distilled H2O for
?. [H&E Staining]
Rinse slides with Bluing solution by moving slides up and down for  to
?. [H&E Staining]
Wash slides with distilled H2O for
?. [H&E Staining]
Stain slides with Eosin for  to
?. [H&E Staining]
Wash/dehydrate slides with 75% ethanol for  to
?. [H&E Staining]
Dehydrate slides with 95% ethanol for  to
?. [H&E Staining]
Dehydrate slides with 100% ethanol for   (twice)
?. [H&E Staining]
Immerse slides in xylene for   (twice)
?. [H&E Staining]
Air dry slides under the hood for  to
?. [H&E Staining]
Mount sections with Permount and cover with coverslips
?. [H&E Staining]
Stained amples are imaged using an Aperio ScanScope XT
?. [H&E Staining]
H&E images are uploaded to METASPACE and registered to the corresponding MSI data
?. [H&E Staining]
Slides are then re-stained with PAS
?. [PAS Staining]
Place the slides in 10% formalin in 95% alcohol for  .
?. [PAS Staining]
Oxidize in 0.5% periodic acid solution for
?. [PAS Staining]
Rinse in tap water four times.
?. [PAS Staining]
Place in Schiff reagent for
Sections become a light pink color
?. [PAS Staining]
Wash in lukewarm tap water for
Sections immediatly become a dark pink color
?. [PAS Staining]
Counterstain in Mayer's hematoxylin for
?. [PAS Staining]
Rinse in TBS solution and then rinse in deionized water four times.
?. [PAS Staining]
Dehydrate by the successive ethanol and xylene steps 14 to 18 and coverslip using a synthetic mounting medium.
?. [PAS Staining]
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.
?. [PAS Staining]
Rinse in tap water four times.