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

• Main Authors: Tan, Yong Chiang, Mustangin, Muaatamarulain, Rosli, Nurwahyuna, Wan Ahmad Kammal, Wan Syahira Ellani, Md Isa, Nurismah, Low, Teck Yew, Lee, Pey Yee, Chellappan, Dinesh Kumar, Jarmin, Razman, Zuhdi, Zamri, Azman, Azlanudin, Ian, Chik, Mohamed Yusof, Nursuhadah, Lim, Lay Cheng
• Format: Article
• Language: English
• Published: Academic Press Inc. 2024
• Online Access: http://psasir.upm.edu.my/id/eprint/115064/1/115064.pdf; http://psasir.upm.edu.my/id/eprint/115064/; https://linkinghub.elsevier.com/retrieve/pii/S0011224023003425
• Institution: Universiti Putra Malaysia

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 < 0.005), while EtOH-LN (SD = 0.56) generates more consistent results compared to LN-only (SD = 1.29). We have modified a previously reported morphology restoration method to incorporate the EtOH-LN workflow, which successfully restored the tissue architecture from freezing artifacts (p < 0.05). Additional studies are required to validate the impact of the EtOH-LN workflow on the molecular profiles of tissues.