New developments in pathological tissue analysis techniques: Immunohistochemical staining with NanoSuit-CLEM (correlative light and electron microscopy)

Abstract

Immunohistochemical (IHC) staining and in situ hybridization coupled with optical microscopy are the primary methods for tissue analyses. When it is necessary to examine finer structures, electron microscopy can be used. This review explains the principles and methods for IHC and scanning electron microscopy (SEM) coupled with NanoSuit- correlative light and electron microscopy (CLEM). Additionally, we present our own cases utilizing p16 IHC, a well-used practice among otolaryngologists.

IHC uses antigen-antibody reactions to stain specific proteins in tissues. The general procedure includes sectioning from a formalin-fixed paraffin-embedded block, antigen retrieval, blocking nonspecific reactions, primary and secondary antibody reactions, color development, and mounting. Establishing optimal conditions for each step can be challenging.

SEM requires high-vacuum conditions for observation, necessitating tissue drying. Various treatments are required to prevent deformation caused by drying. The NanoSuit method allows the observation of live Drosophila larvae by SEM under high vacuum, and facilitates SEM observation by applying a NanoSuit solution without drying the tissue, thus preserving the original structures. Using NanoSuit-CLEM, we successfully observed human papillomavirus (HPV) particles by examining sites of HPV-L1 (capsid protein) IHC positivity and also visualized other viral particles.

NanoSuit-CLEM offers high compatibility with pathological techniques and can be widely applied in various research fields.