Year: 2024
Type: preprint
Abstract: This protocol covers the harvesting, preservation, and implementation of sequential RNA-FISH on human articular cartilage. Distinct elements of this protocol that worked well for human articular cartilage include: (1) the use of Paxgene fixative and stabilizer for the initial preservation of RNA immediately following the initial tissue harvest. (2) the permeabilization of tissue sections for probe access by sequential treatment with a hydrogen peroxide-formamide solution followed by digestion with protease plus, and treatment with HCl. (3) Finally, while the probe design and hybridization chemistry were essentially identical to MERFISH, after covering the tissue in an acrylamide hydrogel, the tissue was not fully cleared by digestion as the loose attachment of chondrocytes sitting inside their lacunae would be lost. Thus, the imaging procedure first captured background matrix autofluorescence, which in the image processing pipeline was background subtracted. Collectively, this protocol worked well on articular cartilage samples derived from 6 different human donors of varying age and ancestry.
Authors: Peter Maye, Mejeong Lee, David Rowe, Martin Lotz, Merissa Olmer, dong.shin not provided
Institutions: UConn Health, University of Connecticut
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W4409561032