Novel method of paraffin embedding cultured cells and organoids using silicone molds.

Abstract

A major issue facing the field of cellular imaging, immunofluorescence (IF), and immunohistochemistry (IHC) microscopy is antibody quality. One of the main methods of antibody validation is testing on positive and negative control tissues with known expression levels of a given antigen. However, this approach is reliant on availability of tissues and reliable protein expression datasets, which are not always available. In contrast, cultured cell lines often have more extensive and reproducible protein expression data available, are relatively inexpensive to maintain, and can be used to produce knockout lines for more robust and functional validation. Due to the difference in staining protocols between formalin-fixed paraffin-embedded (FFPE) tissues and cultured cell lines, an antibody that works well in cultured cells does not always produce the same results in FFPE tissues. For this reason, there is a need for methods to embed cultured cells in paraffin for antibody testing. Previous methods have been published, but many involve use of sharps, which introduces risk of cuts to the investigator, or embedded in agarose first, which results in a lower density of cells. This paper introduces a method of embedding cultured cells using custom designed silicone molds. These molds allow an easy, risk-free embedding process that results in high density cell pellet blocks which can be used for IF and IHC experiments, as well as creation of cell microarrays. Additionally, the silicone molds can be used to embed organoids for IF and IHC analysis.