Reversing the Effects of Formalin Fixation with Citraconic Anhydride and Heat: A Universal Antigen Retrieval Method

Abstract

Formalin is a commonly used fixative for tissue preservation in pathology laboratories. A major adverse effect of this fixative is the concealing of tissue antigens by protein cross-linking. To achieve a universal antigen retrieval method for immunohistochemistry under a constant condition, we developed a new method in which the effects of formalin fixation were reversed with citraconic anhydride (a reversible protein cross-linking agent) plus heating. Formalin-fixed, paraffin-embedded tissues from various organs were examined for immunohistochemical localization of a wide variety of antigens. Deparaffinized tissue sections were placed in an electric kitchen pot containing 0.05% citraconic anhydride solution, pH 7.4, and the pot was set at "keep warm" temperature mode of 98C for 45 min. This mode allowed heating the sections at a constant temperature. The sections were then washed in buffer solution and immunostained using a labeled streptavidin-biotin method using an automated stainer. In general, formalin-fixed tissues demonstrated specific immunostainings comparable to that in fresh frozen tissues and significantly more enhanced than after conventional antigen retrieval methods. In particular, even difficult-to-detect antigens such as CD4, cyclin D1, granzyme beta, bcl-6, CD25, and lambda chain revealed distinct immunostainings. Different classes of antigens such as cellular markers and receptors, as well as cytoplasmic and nuclear proteins, consistently produced enhanced reactions. This method provides efficient antigen retrieval for successful immunostaining of a wide variety of antigens under an optimized condition. It also allows standardization of immunohistochemistry for formalin-fixed tissues in pathology laboratories, eliminating inter-laboratory discrepancies in results for accurate clinical and research studies.

A nondestructive molecule extraction method allowing morphological and molecular analyses using a single tissue section

“…Recently, there is increasing evidence that formalin-induced macromolecule crosslinking can be reversed under high temperature. [29][30][31] Heating treatment not only transformed intermolecular cross-linked polymers into monomers 24,29,30 but also partially restored enzymatic reactivity of formalinfixed RNase A. 31 Based on literature and our observations, we believe that heating effect, either direct heating as described herein or microwave/ ultrasound-induced heating, plays a pivotal role in the NDME procedures for FFPE tissues.…”

Folate receptor 1 is necessary for neural plate cell apical constriction during Xenopus neural tube formation

“…Primary antibodies used were: anti-Folr1 (custom-made by GenScript; 1:400), anti-C-cadherin (6B6, Developmental Studies Hybridoma Bank; 1:50), anti-β-tubulin (E7, Developmental Studies Hybridoma Bank; 1:300), anti-E-cadherin (5D3, Developmental Studies Hybridoma Bank; 1:100), anti-GFP (GFP-1020, Aves Labs; 1:500), anti-Sox2 (AF2018, R&D Systems; 1:300), anti-m-Cherry (ab 167453, Abcam; 1:500). Antigen retrieval was performed by microwaving samples in 0.05% citraconic anhydride, pH 7.4 (Namimatsu et al, 2005). Briefly, a rack with slides was placed in a 200 ml glass container covered with plastic wrap, boiled for 15 s at maximum power (1200 W) followed by 3 min wait in hot buffer.…”

‘Tissue surrogates' as a model for archival formalin-fixed paraffin-embedded tissues

“…Solutions of 6 M guanidine HCL with BME 12 and aqueous 0.05% citraconic anhydride 13 were also evaluated. The surrogates were then homogenized with a disposable pellet pestle (Kontes Scientific, Vineland, NJ, USA), followed by two 10-s cycles of sonication on ice using a Sonic Dismembrator, model 550, fitted with a 0.125-inch tapered microtip (Fisher Scientific).…”

“…These results are shown in Table 4. Namimatsu et al 13 reported improved immunohistochemical staining of Tissue surrogates heated in 6 M guanidine HCl supplemented with 0.5 M BME, a disulfide-reducing agent, resulted in a protein recovery of 58%. Recovery efficiency was increased to 470% in solutions of 20 mM Tris HCl with 2% SDS and 0.5 M BME (Table 4).…”