Antigen Retrieval Causes Protein Unfolding

Abstract

Formalin fixation and paraffin embedding (FFPE) remains the preeminent technique for processing tissue specimens for pathologic examination, for the study of tissue morphology, and for archival preservation (Fox et al. 1985). The steps of FFPE tissue processing are generally as follows. Following fixation in a 3.7% solution of neutral-buffered formalin (fixation), tissue specimens are dehydrated through a series of solutions with increasing concentrations of alcohol (graded alcohols). Samples are then soaked in a transition solvent such as xylene (cleared) followed by liquid paraffin, which is then cooled for long-term storage as solid blocks that can be cut into thin slices for mounting on slides for microscopy.The early work of Fraenkel-Conrat (Fraenkel-Conrat et al. 1947;Fraenkel-Conrat and Ollcott 1948;Fraenkel-Conrat and Mecham 1949) and the more recent work of Metz et al. (2004Metz et al. ( , 2006 have identified four types of chemical modifications following treatment of peptides or proteins with formaldehyde. These modifications are as follows: hydroxymethyl (methylol) adducts, Schiff base adducts, 4-imidazolidinone adducts, and methylene bridges (cross-links). Methylol and Schiff base adducts form rapidly upon reaction with primary amino (lysine) and thiol (cysteine) groups. The SummaryAntigen retrieval (AR), in which formalin-fixed paraffin-embedded tissue sections are briefly heated in buffers at high temperature, often greatly improves immunohistochemical staining. An important unresolved question regarding AR is how formalin treatment affects the conformation of protein epitopes and how heating unmasks these epitopes for subsequent antibody binding. The objective of the current study was to use model proteins to determine the effect of formalin treatment on protein conformation and thermal stability in relation to the mechanism of AR. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to identify the presence of protein formaldehyde cross-links, and circular dichroism spectropolarimetry was used to determine the effect of formalin treatment and high-temperature incubation on the secondary and tertiary structure of the model proteins. Results revealed that for some proteins, formalin treatment left the native protein conformation unaltered, whereas for others, formalin denatured tertiary structure, yielding a molten globule protein. In either case, heating to temperatures used in AR methods led to irreversible protein unfolding, which supports a linear epitope model of recovered protein immunoreactivity. Consequently, the core mechanism of AR likely centers on the restoration of normal protein chemical composition coupled with improved accessibility to linear epitopes through protein unfolding. (J Histochem Cytochem 59:366-381, 2011)

Paraffin Immunofluorescence: A Valuable Ancillary Technique in Renal Pathology

“…In contrast to IF-F, which is performed on cryostat sections cut from unfixed frozen tissue, IF-P is performed on formalin-fixed, paraffin-embedded tissue. Because formalin fixation induces protein cross-linking, which generally blocks antigenicity, an antigen-retrieval step that allows for increased penetration of antibodies to the antigens “masked” by formalin fixation is required in IF-P. 5 , 6 This step involves incubating the paraffin sections with a proteolytic enzyme or heating the sections before incubation with fluorescein isothiocyanate–conjugated antibodies against Igs and complement components. Multiple proteolytic enzymes have been used in IF-P, including trypsin, 7 , 8 , 9 , 10 , 11 , 12 pronase E (protease XIV), 13 , 14 , 15 , 16 , 17 proteinase XXIV, 18 and proteinase K. 13 , 19 , 20 , 21 , 22 Successful results were also obtained by heat treatment with Tris or citrate buffers 15 and with dual microwave heating in EDTA antigen-retrieval solution.…”

A Secondary Antibody-Detecting Molecular Weight Marker with Mouse and Rabbit IgG Fc Linear Epitopes for Western Blot Analysis

“…Antibodies are widely used in several types of immunoassays such as immunoblotting [ 18 , 19 ], immunohistochemistry (IHC) [ 14 ], immunocytochemistry [ 20 ], immunoprecipitation (IP) [ 21 ], enzyme-linked immunosorbent assay (ELISA) [ 22 , 23 ] and fluorescent in situ hybridization [ 24 , 25 ]. Because mice and rabbits are the most commonly used hosts for immunization and primary antibody production [ 24 ], commercial secondary antibodies that recognize mouse and rabbit antibodies are widely available [ 26 ].…”

A technical review and guide to RNA fluorescence in situ hybridization

“…To fix tissue sections or whole-mounts, alcohol is commonly combined with other fixatives such as formaldehyde (Finnerty et al, 2003;Martindale, Pang & Finnerty, 2004;Hejnol & Martindale, 2008;Pearson et al, 2009). Although methanol has been used successfully with immunofluorescence (Levitt & King, 1987), methanol has a propensity to disrupt native protein structure and is generally not recommended for use in multiplex FISH and immunohistochemistry (Fowler et al, 2011). Methanol will strip membrane lipids to improve permeability (Hoetelmans et al, 2001) and ethanol can strip the external wax and lipids from plant tissues (Bleckmann & Dresselhaus, 2016).…”

“…With the use of formaldehyde, heat can accelerate the fixation process; although heat also increases the release of formaldehyde fumes which are hazardous to human health (Fox et al, 1985;Titford, 2001). Additionally, heat can denature proteins and cause a loss of antigenicity which would negatively affect multiplex FISH and immunohistochemistry (Fowler et al, 2011). For nucleic acid visualization, reduced temperatures of 4 C have been shown to preserve RNA throughout the fixation process (Bussolati et al, 2011).…”