A technique for retrieving antigens in formalin-fixed, routinely acid-decalcified, celloidin-embedded human temporal bone sections for immunohistochemistry.

Abstract

The application of immunohistochemistry to routinely decalcified, celloidin-embedded human temporal bone sections has been hampered because of antigen loss during processing of the specimens. To our knowledge, there has been no published report to date describing immunohistochemical staining of such tissues suitable for examination by light microscopy. Here we report a novel antigen retrieval technique which can be successfully used to stain a variety of antigens in routinely formalin-fied, trichloroacetic acid-decalcified, celloidin-embedded human temporal bone sections. The new procedure reported here for decalcified human temporal bone tissues simply requires immersing slides for 30 min at room temperature in an antigen retrieval solution. A total of 60 decalcified, celloidin-embedded human temporal bone tissues were tested with monoclonal antibodies (MAb) to 15 different antigens. Of these, 12 MAb showed defiite positive staining, while three were negative. This technique may prove very useful in studying the expression of various antigens by immunohistochemistry in formalin-fiied, aciddecalcified, celloidin-embedded tissues. (J Hisrochem Cytochem 40:787-792, 1992)

Antigen Retrieval Immunohistochemistry: Past, Present, and Future

“…It has been applied for a series of immunohistochemical studies on human temporal bone sections (Linthicum et al 1995). Recently, Harkins and Grizzle (Workshop presentation;1995 Natl Soc Histotech Ann Symp) found that the use of the NaONmethanol solution (Decal; BioGenex, San Ramon, CA) could improve immunostaining of keratin, vimentin, GFAP, desmin, muscle specific actin, S-100, NSE, kappa, lambda, bcl-2, B-cells (L-26), and T-cells (UCHL) in formalin-fixed, acid, or EDTA-decalcified, paraffin-embedded tissues, in addition to celloidinembedded tissues reported previously (Shi et al 1992a(Shi et al ,b,1993a. Perhaps, there may be a simple method for non-heating AR by combining the use of acid and alkaline solutions, such as a "hydrolysis" procedure.…”

“…First of all, it showed that the modification of protein structure by formalin is reversible under certain conditions, such as high-temperature heating or strong alkaline treatment. We have suggested a possible mechanism of the AR technique, i.e., loosening or breaking of the crosslinkages caused by formalin fixation (Shi et al 1991(Shi et al ,1992a. A recent study by Mason and O'Leary (1991) has demonstrated that the process of crosslinking does not result in discernible alteration of protein secondary stucture.…”

“…Biochemical studies of the chemical reaction between protein and formalin by Fraenkel-Conrat and co-workers in the 1940s (1947,1948a,b) indicated that hydrolysis of crosslinkages between formalin and protein is limited by certain amino acid side chains, such as imidazol and indol, but that these crosslinkages can be reversed by high-temperature heating (120C) or strong alkaline treatment. This observation formed the basis for the development of AR techniques in 1991 (Shi et al 1991(Shi et al ,1992a. A simple method of either heating the routinely fixed paraffin-embedded tissue sections in water or immersing the routinely formalin-fixed, aciddecalcified, celloidin-embedded tissue sections in an NaOH-methanol solution yielded dramatic retrieval results (Shi et al 1991(Shi et al ,1992a(Shi et al ,b,1993a.…”

“…This observation formed the basis for the development of AR techniques in 1991 (Shi et al 1991(Shi et al ,1992a. A simple method of either heating the routinely fixed paraffin-embedded tissue sections in water or immersing the routinely formalin-fixed, aciddecalcified, celloidin-embedded tissue sections in an NaOH-methanol solution yielded dramatic retrieval results (Shi et al 1991(Shi et al ,1992a(Shi et al ,b,1993a. The AR technique has been used increasingly and has demonstrated its value in many studies for over 200 antibodies Werner et al 1996;Boon and Kok 1995;Brown and Chirala 1995;Cattoretti and Suumeijer 1995;Shi et al 1995b;Cuevas et al 1994;Gu 1994;Gu et al 1994a,b;Igarashi et al 1994;Kawai et al 1994a;Swanson 1994;Taylor and Cote 1994;Cattoretti et al 1993;Gown et al 1993a;Leong and Milios 1993;McKee et al 1993;Norton 1993;Suurmeijer and Boon 1993a,b;Shi et al 1991).…”

Enhanced Immunohistochemical Detection of Infectious Agents in Formalin-Fixed, Paraffin-Embedded Tissues following Heat-Mediated Antigen Retrieval

“…6 In the past decade, other methods to restore reactive sites in fixed tissues have been described. 1,7,10 A recently described method is heat-mediated antigen retrieval (HMAR). This technique has been useful for enhancing the immunohistochemical detection of a number of antigens.…”