Tissue fixation is the first of many critical steps in the successful preparation of histological sections and stained slides. Fixation terminates ongoing biological reactions (including decay) whilst maintaining excellent morphological tissue microstructure. One of the most widely used methods of tissue preservation within our laboratory is formaldehyde fixation. This fixative works by cross-linking amino acids and other functional groups within proteins throughout the sample.
Antigen retrieval (AR) is the process of breaking or reversing these chemical bonds to allow access to the desired protein target antigen of choice. It is an essential step in IHC protocol development as - without successful AR, reagents will not have adequate access to the antigen and resultant staining will be unsatisfactory. A wide variety of techniques and instruments have been developed in recent years to standardise AR and to help address the loss of antibody sensitivity as a result of formaldehyde fixation (leading to unsatisfactory staining). Two common examples of AR include enzymatic digestion and heat induced epitope retrieval (HIER). During IHC protocol development, the selection of AR method has largely remained an empirical process. The ultimate goal is to utilise a suitable AR method to allow clean, specific antigen staining without damaging the morphology of the tissue.
In this study, Vollert et al. examined the chemistry of formaldehyde fixation and AR by measuring formaldehyde adduct formation on two model peptides using MALDI-TOF mass spectrometry. Formaldehyde scavenging agents were later introduced as alternative AR reagents.
There are 3 types of formaldehyde chemical modification that occur during fixation. 1) Methylol groups, 2) Schiff bases and 3) Methylene bridges (Typically only seen during long-term over-fixation). Types 1 and 2 are the primary adducts associated with routine formaldehyde fixation. These adducts are reversible and are therefore the main targets of AR.
Angiotensin I and adrenocorticotropic hormone (ACHT) were chosen as model peptides due to their multiple binding sites for formaldehyde and potential adduct formation. When treated solely with formaldehyde, complete conversion to mono and di-formyl adducts was seen – demonstrating desired fixation. Following post-fixation heating (as in standard HIER protocol), conversion of the di-formyl group back to the mono and (in low levels) conversion back to the original Angiotensin I peptide was seen. This suggests formaldehyde adduct formation is reversible with heat.
Significant regeneration of Angiotensin I was seen at pH 3 but not at pH 7 or pH 10. It was suggested that formaldehyde scavenging agents could aid AR by binding to liberated formaldehyde in solution. This scavenging may prevent protein-dissociated formaldehyde molecules from re-attaching to other open binding sites on tissues thus increasing the effectiveness of formaldehyde dissociation during the AR process.
These findings were translated to FFPE sections of mouse brain where the use of formaldehyde scavenging agents as AR regents were compared to traditional AR reagents (sodium citrate) in their effectiveness in providing formaldehyde sensitive Collagen IV basement membrane labelling.
At optimal pH 3, traditional sodium citrate AR was ineffective and resulted in poor staining. The formaldehyde scavenging agents performed well as novel AR reagents and allowed the effective retrieval of the antigen via the elimination of additional formaldehyde adducts once initially liberated. This resulted in the previously unachievable staining of Collagen IV in FFPE mouse brain and also allowed the robust detection of other vascular antigens that have previously been irretrievably masked by formaldehyde fixation.
This work provides a clear basis for the use of heat and pH in AR and introduces a new approach to identify new and effective AR reagents based on their ability to scavenge previously dissociated formaldehyde.
Vollert et al. 2015 Formaldehyde scavengers function as novel antigen retrieval agents. Scientific Reports volume 5, Article number: 17322
Nature: Scientific Reports https://www.nature.com/articles/srep17322