Immunocytochemistry of formalin-fixed human brain tissues: microwave irradiation of free-floating sections

• Published in: Brain research. Brain research protocols Vol. 2; no. 2; pp. 109 - 119
• Main Authors: Shiurba, Robert A, Spooner, Edward T, Ishiguro, Koichi, Takahashi, Miho, Yoshida, Rie, Wheelock, Timothy R, Imahori, Kazutomo, Cataldo, Anne M, Nixon, Ralph A
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 1998
• Subjects: Brain - anatomy & histology; Brain - radiation effects; Fluorescent Dyes; Formaldehyde; Humans; Immunocytochemistry; Immunohistochemistry - methods; Indicators and Reagents; Microtomy - methods; Microwave antigen retrieval; Microwaves; Neuropathology; Tau protein kinase I/glycogen synthase kinase-3 β; Thiazoles; Tissue Fixation - methods; Tau protein kinase I/glycogen synthase kinase-3 β; Microwave antigen retrieval; Immunocytochemistry; Neuropathology
• ISSN: 1385-299X
• DOI: 10.1016/S1385-299X(97)00029-9

Formalin fixation, the chemical process in which formaldehyde binds to cells and tissues, is widely used to preserve human brain specimens from autolytic decomposition [10, 16, 25, 41]. Ultrastructure of cellular and mitochondrial membranes is markedly altered by vesiculation [16], but this does not interfere with diagnostic evaluation of neurohistology by light microscopy. Serious difficulties are encountered, however, when immunocytochemical staining is attempted. Antigens that are immunoreactive in unfixed frozen sections and protein extracts appear to be concealed or destroyed in formalin-fixed tissues. In dilute aqueous solution, formaldehyde is in equilibrium with methylene glycol and its polymeric hydrates, the balance by far in favor of methylene glycol [16, 41]. Carbonylic formaldehyde is a reactive electrophilic species well known for crosslinking functional groups in tissue proteins, nucleic acids, and polysaccharides [16–20, 22, 27]. Some of its methylene crosslinks are readily hydrolyzed. Others are stable and irreversible. During immunostaining reactions, intra- and inter-molecular links between macromolecules limit antibody permeation of tissue sections [16], alter protein secondary structure [26, 34], and reduce accessibility of antigenic determinants [10]. Accordingly, immunoreactivity is diminished for many antigens. Tissues are rapidly penetrated by methylene glycol, but formaldehyde binding to cellular constituents is relatively slow, increasing progressively until equilibrium is reached [16, 24]. In addition, prolonged storage in formalin may result in acidification of human brain specimens [42]. Low pH favors dissociation of methylene glycol into formaldehyde [16], further reducing both classical staining and antigen detectability [14, 42]. Various procedures have been devised to counter the antigen masking effects of formaldehyde. Examples include pretreatment of tissue sections with proteases [2, 7, 23], formic acid [3, 28], or ultrasound [43]. Recently, heating of mounted sections in ionic salt solution by microwave energy was found to restore many antigens [6, 37, 44–48, 54–56]. Theory and practice of microwave antigen retrieval are covered extensively in the handbook Microwave Cookbook for Microscopists [29]. A concise overview of microwave methods in the neurosciences has been published [33], and clinical applications have been reviewed [30]. In this context, it should be noted that fresh tissues may be stabilized for immunocytochemistry by reversible, non-chemical binding processes such as cryosectioning after microwave treatment [32]and freeze-drying [52]. Thus, it may be possible to enhance immunostaining for some antigens by microwave irradiation of unfixed as well as fixed specimens. Parameters to be optimized for microwave retrieval of specific antigens include temperature, irradiation time, tissue buffer composition, salt concentration, and pH [12, 47, 53]. Temperature, irradiation time, and pH are key variables [12, 13]. With this in mind, an optimal method was developed for retrieval of a wide variety of antigens in human brain tissues [14]. Typical microwave protocols employ elevated temperatures that may reach 100°C, where denaturation causes irreversible uncoiling and disruption of protein secondary and tertiary structures [11]. Under these conditions, stable covalent bonds securing methylene crosslinks between polypeptides remain intact [20], but more reactive links formed by Schiff bases may be hydrolyzed [45]. Resultant conformational changes presumably expose buried loops of continuous amino acids and protruding regions [1], increasing accessibility of their epitopes [51]. Protein denaturation seems to be a reasonable explanation for the effects of microwaves on antigen retrieval. This idea is supported by the observation that denaturing solutions such as 6 M urea increase immunoreactivity of some antigens [8]. Still, the molecular basis of these effects remains unresolved, in part due to the complex chemistry of formaldehyde reactions with tissue constituents [41]. Indeed, some methylene bridges between similar groups such as NH 2 and NH may be hydrolyzed by washing fixed tissues in distilled water at ambient temperature for several weeks [24, 41]. Moreover, denaturation by conventional heating enhances antigenicity as well as classical neuroanatomical staining of formalin-fixed tissue [15, 39, 40, 49]. When such externally heated specimens are immunostained and viewed by light microscopy, the results are almost indistinguishable from those obtained by microwave irradiation [58]. Nevertheless, the current widespread use of microwave methods in clinical and basic science laboratories likely results from the speed, convenience, and reproducibility of the results. Loss of immunoreactivity for many antigens likewise may occur when tissues are dehydrated in alcohol before they are embedded in paraffin. Exposure to alcohol causes antigenic denaturation, but clearing in xylene and heating in liquid paraffin do not [52]. While there are numerous reports of microwave procedures for formalin-fixed, paraffin-embedded tissues [6, 8, 9, 35, 36, 38, 44, 45, 48, 57, 60], alternative methods that would further extend the range of retrievable epitopes have received less attention. One simple approach may be to irradiate free-floating sections with microwaves. A previous study found that heating vibratome sections in solution with microwaves resulted in severe wrinkling of the tissues. This problem was avoided by irradiation of tissue slices in buffer prior to sectioning [13]. Another strategy is presented here for neuropathological studies of human brain [50]. Vibratome sections in isotonic, mildly acidic citrate buffer [6, 55]are heated to the boiling point with microwaves. After brief denaturation at 100°C, the sections are simmered for 5 min. To remove wrinkles, sections are incubated subsequently in Tris-buffered saline containing serum proteins and non-ionic detergent. The method also is suitable for single- and double-labeling studies of neural antigens in formalin-fixed tissues from experimental animals.