AUTHOR=Muniz Partida Carolina , Walters Eric 

TITLE=A novel immunohistochemical protocol for paraffin embedded tissue sections using free-floating techniques

JOURNAL=Frontiers in Neuroanatomy

VOLUME=Volume 17 - 2023

YEAR=2023

URL=https://www.frontiersin.org/journals/neuroanatomy/articles/10.3389/fnana.2023.1154568

DOI=10.3389/fnana.2023.1154568

ISSN=1662-5129

ABSTRACT=Immunohistochemistry (IHC) is a well-established and widely used protocol used to visualize tissue architecture, protein expression and localization. Free-floating methods for IHC employ tissue sections that are cut from a cryostat or vibratome. The limitations of these tissue sections are tissue fragility, poor morphology, and the need to use sections of 20-50 um. In addition, there is a void of information regarding the use of free floating immunohistochemical techniques on paraffin embedded tissue. To address this, we developed a free-float IHC protocol with paraffin embedded tissue (PFFP) that saves time, resources, and tissues. PFFP localized GFAP, OMP, Nestin, GAP43 expression in mouse hippocampal, olfactory bulb, striatum, and cortical tissue. Successful localization of these antigens was achieved using PFFP with and without antigen retrieval, with subsequent chromogenic DAB (3,3’-Diaminobenzidine) development and immunofluorescence detection methods. Furthermore, PFFP allowed for the detection of antigens in as little as four hours. The application of the PFFP in combination with other methods such as in situ hybridization, studying protein / protein interactions, laser capture dissection and pathological diagnosis expands the versatility of paraffin embedded tissues.