EtOH-LN cryoembedding workflow to minimize freezing artifact in frozen tissues: A pilot study in preparing tissues compatible with mass spectrometry-based spatial proteomics application

Tan YC 1 , Mustangin M 2 , Rosli N 3 , Wan Ahmad Kammal WSE 4 , Md Isa N 5 , Low TY 6 Show all authors , Lee PY 7 , Chellappan DK 8 , Jarmin R 9 , Zuhdi Z 10 , Azman A 11 , Ian C 12 , Yusof NM 13 , Lim LC 14

Affiliations 

  • 1 School of Postgraduate Studies, International Medical University, Kuala Lumpur, Malaysia. Electronic address: yongchiangtan@imu.edu.my
  • 2 Department of Pathology, UKM Medical Centre (UKMMC), Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia. Electronic address: amar@ppukm.ukm.edu.my
  • 3 Department of Pathology, UKM Medical Centre (UKMMC), Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia. Electronic address: nurwahyuna@ukm.edu.my
  • 4 Diagnostic Laboratorial Services Department, UKM Medical Centre (UKMMC), Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia. Electronic address: ellani@ppukm.ukm.edu.my
  • 5 Department of Pathology, UKM Medical Centre (UKMMC), Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia. Electronic address: nuris@ppukm.ukm.edu.my
  • 6 UKM Medical Molecular Biology Institute (UMBI), Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia. Electronic address: lowteckyew@ppukm.ukm.edu.my
  • 7 UKM Medical Molecular Biology Institute (UMBI), Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia. Electronic address: leepeyyee@ppukm.ukm.edu.my
  • 8 Department of Life Sciences, School of Pharmacy, International Medical University, Kuala Lumpur, Malaysia. Electronic address: dinesh_kumar@imu.edu.my
  • 9 Department of Surgery, UKM Medical Centre (UKMMC), Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia. Electronic address: razman@ppukm.ukm.edu.my
  • 10 Department of Surgery, UKM Medical Centre (UKMMC), Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia. Electronic address: zamriz7582@gmail.com
  • 11 Department of Surgery, UKM Medical Centre (UKMMC), Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia. Electronic address: drazlan@ppukm.ukm.my
  • 12 Department of Surgery, UKM Medical Centre (UKMMC), Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia. Electronic address: ianchik@ukm.edu.my
  • 13 Department of Surgery, UKM Medical Centre (UKMMC), Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia. Electronic address: nursuhadah@ppukm.ukm.edu.my
  • 14 Department of Life Sciences, School of Pharmacy, International Medical University, Kuala Lumpur, Malaysia. Electronic address: laychenglim@imu.edu.my
Cryobiology, 2024 Mar;114:104843.
PMID: 38158171 DOI: 10.1016/j.cryobiol.2023.104843

Abstract

Coolant-assisted liquid nitrogen (LN) flash freezing of frozen tissues has been widely adopted to preserve tissue morphology for histopathological annotations in mass spectrometry-based spatial proteomics techniques. However, existing coolants pose health risks upon inhalation and are expensive. To overcome this challenge, we present our pilot study by introducing the EtOH-LN workflow, which demonstrates the feasibility of using 95 % ethanol as a safer and easily accessible alternative to existing coolants for LN-based cryoembedding of frozen tissues. Our study reveals that both the EtOH-LN and LN-only cryoembedding workflows exhibit significantly reduced freezing artifacts compared to cryoembedding in cryostat (p 

* Title and MeSH Headings from MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

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