Optimization of Whole-Body Zebrafish Sectioning Methods for Mass Spectrometry Imaging

Abstract

Mass spectrometry imaging (MSI) methods and protocols have become widely adapted to a variety of tissues and species. However, the MSI literature contains minimal information on whole-body cryosection preparation for the zebrafish (ZF; Danio rerio), a model organism routinely used in developmental, toxicity, and carcinogenicity studies. The optimal medium for embedding and cryosectioning a whole organism or soft-tissue specimen for histological examination is a synthetic polymer mixture that is incompatible with MSI as a result of ion suppression. We describe the optimal methods and results for embedding and cryosectioning whole-body ZF for MALDI-MSI. We evaluated 13 distinct embedding media formulations and found a supportive hydrogel with the consistency of cartilage to be the optimal embedding medium. The hydrogel medium does not interfere with MSI data collection, aids in tissue stability, is readily available for purchase, and is easy to prepare and handle during cryosectioning. Additionally, we decreased the matrix cluster interference commonly caused by α-cyano-4-hydroxycinnamic acid by adding ammonium phosphate to the solvent spray solution. The optimized methods developed in our laboratory produced high-quality cryosections, as well as high-quality mass spectral images of sectioned ZF.

Use of whole‐body cryosectioning and desorption electrospray ionization mass spectrometry imaging to visualize alkaloid distribution in poison frogs

“…This was the only medium that was not too hard to tear the section, disintegrating the tissues and hindering the histological analysis, but hard enough to maintain rigidity at a proper temperature. The solution was also the most pliable and stable medium for frog cross‐sectioning, in agreement with similar results found for zebrafish cryosectioning 28 . The minimum section thickness we achieved was 15 μm.…”

“…We tested three different embedding media: 2% carboxymethyl cellulose (CMC), 5% CMC, and 5% CMC + 10% gelatin based on procedures described for zebrafish by Nelson et al 28 and Perez et al 29 We removed the arms and legs of the frogs 7 , placed the whole body in a flexible, peel‐away mold with embedding media, and oriented it to sagittal sectioning. To visualize simultaneously as many internal organs as possible, we analyzed three regions of the body through mid‐sagittal, left, and right parasagittal sections.…”

Imaging of whole zebra fish (Danio rerio) by desorption electrospray ionization mass spectrometry

“…Slices were taken at this thickness due to the ease of reproducibility with the chosen embedding medium. A selection of embedding media has previously been studied by Nelson et al . for the purpose of freezing and cryosectioning zebra fish.…”

“…Slices were taken at this thickness due to the ease of reproducibility with the chosen embedding medium. A selection of embedding media has previously been studied by Nelson et al [21] for the purpose of freezing and cryosectioning zebra fish. They determined that a more optimal medium consisting of 5% CMC and 10% gelatine mixture can be used to achieve reproducible slices of 16 μm thickness.…”

Improved spectra for MALDI MSI of peptides using ammonium phosphate monobasic in MALDI matrix

“…Lagarrigue et al dipped the slides in ice‐cold 10 mM AmP after matrix application for suppressing α‐CHCA matrix cluster formation . In the same concept, Nelson et al investigated matrix application conditions specifically for peptide and protein detection in whole‐body zebrafish by MALDI MSI . The writers showed for the first time AmP (6 mM AmP in 5 mg/mL α‐CHCA) usage with an automatic sprayer and reported that AmP in MALDI matrix enhanced analyte intensities.…”