Fresh-frozen lenses were mounted on cold chucks using Cryomatrix compound (Thermo Scientific) at the base of the tissue only. Equatorial or axial sections (20 μm) were cut using a cryostat (Leica CM3050S, Leica Microsystems GmbH, Wetzlar, Germany). Bovine lens sections were methanol soft landed30 (link) onto gold-coated MALDI targets while human lenses were thaw mounted onto polylysine-coated ITO glass slides (Delta Technologies, Loveland, CO, USA), polylysine coated in house. Sections were then vacuum desiccated for 30 minutes prior to tissue washing for crystallin protein signal enhancement.
Cryomatrix compound
Manufactured by Thermo Fisher Scientific
Sourced in Belgium, United States
Cryomatrix compound is a ready-to-use, embedding medium for snap-freezing tissue samples for cryosectioning. It is designed to provide a stable support for tissue specimens during the freezing process and subsequent sectioning.
Automatically generated - may contain errors
3 protocols using cryomatrix compound
1
Cryogenic Lens Sectioning and Preparation
2
Cryo-Sectioning and Laser Capture Microdissection of Tongue Tissues
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Tongues were snap-frozen in dry ice-cooled 2-methylbutane (Acros, Geel, Belgium), and embedded in cryomatrix compound (Thermo Fisher Scientific, Waltham, MA). A Leica CM 3050S (Leica Microsystems, GmbH, Nussloch, Germany) cryostat with installed CryoJane®, was used for cryosectioning. Frozen 7 μm tissue sections were mounted onto commercial CryoJane® glass slides. An Arcturus® laser capture microscope (Thermo Fisher Scientific) was used to retrieve epithelial tissue from tongue dorsal and ventral surfaces. Samples were pooled and solubilized in Cell Lysate Buffer® (Signosis, Sunnyvale, CA) for direct reverse transcription, and relative cDNA levels were quantified by RT-qPCR, as described above.
3
Cryosectioning and Matrix Deposition for Mass Spectrometry Imaging
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Frozen lenses were mounted on cold chucks using Cryomatrix™ compound (ThermoFisher Scientific, Waltham, MA, United States). A cryostat (Leica CM3050S, Leica Microsystems GmbH, Wetzlar, Germany) was used to cut axial sections with a thickness of 20 μm. Sections were then immediately mounted on cooled double-sided carbon tape (ProSciTech, Kirwan, Australia) attached to non-conductive glass slides (PINK COLORFROST, LabServ, NZ). Collected sections were stored in a vacuum desiccator for at least 1 h and equilibrated to room temperature immediately before the application of the matrix. A N-(1-naphthyl) ethylenediamine dihydrochloride (NEDC) solution (7 mg/mL in 90% EtOH) containing an internal standard (IS) 3-OMG [(M + Cl)− = m/z 229.0473] was applied using a TM-Sprayer (HTX Technologies, Carrboro, NC). Following matrix/IS spraying, slides were stored in a vacuum desiccator until used for data acquisition (Zahraei et al., 2020 (link)).
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