Effects of antigen retrieval by microwave heating in formalin-fixed tissue sections on a broad panel of antibodies

Abstract

Formaldehyde fixation of biopsy specimens for routine purposes has often been held responsible for the poor reproducibility of immunohistochemical studies. Recently, antigen retrieval (AGR) using microwave irradiation was described as a potential tool to enhance immunostaining. A comparison of conventional staining and staining after microwave heating was performed for 52 markers, using tissues fixed in formaldehyde for 24 h, 1 to 6 weeks and 3 years respectively, as well as consultant case material. After adequate duration of fixation (24 h), only a few markers (17%) showed better results after AGR, but this percentage was increased to 50% when tissues were fixed for longer periods. Maximal enhancement was obtained in the group of consultant cases (58% of tested markers demonstrated better staining results), in which the period of fixation and tissue processing was unknown. To achieve reliable enhancement with AGR, continuous heating (100 degrees C) should not be shorter than 20 min. In conclusion, AGR may become the most important tool to simplify and equalize immunohistochemical techniques, if critically evaluated.

Predominance of the basal type and HER-2/neu type in brain metastasis from breast cancer

“…In brief, sections were deparaffined in xylene, rehydrated, and epitope retrieval was performed in a microwave oven, as described previously. 15 Depending on detection, chromogen endogenous peroxidase or alkaline phosphatase was blocked, and primary antibody was incubated at 41C overnight. The primary antibodies, dilution, and detection method are listed in Table 1.…”

Antigen Retrieval Immunohistochemistry: Past, Present, and Future

“…As mentioned above, a variety of modified fixatives and processing procedures have been used in an attempt to standardize IHC, but have failed. The AR technique can be used to help standardize IHC on archival paraffin sections (Deluque and Luque 1995;von Wasielewski et al 1994;Taylor 1994a;Suurmeijer and Boon 1993a,b;Swanson 1993). On the basis of the maximal retrieval level, it may also be possible to equalize the immunoreactivity in a variety of archival paraffin tissue sections that are fixed in formalin for various periods of time.…”

“…This is a critical issue in interpreting the results of AR-IHC. Thus far, most studies have reported satisfactory results, without false positivity, for a variety of antibodies tested (Brown and Chirala 1995;Lavieille et al 1995;Cuevas et al 1994;Igarashi et al 1994;Taylor et al 1994a;Tenaud et al 1994;von Wasielewski et al 1994;Cattoretti et al 1993;Dookhan et al 1993;Gown et al 1993a;Leong and Milios 1993;Shi et al 1991). Most recently, Baas et al (1996) questioned whether a potential false-positive result of AR-IHC using the MAb DO7 might be obtained after extreme AR treatment.…”

Primary melanoma tumour regression associated with an immune response to the tumour‐associated antigen melan‐A/MART‐1

“…Prior to immunostaining, antigen retrieval was performed by microwaving (800 W; Toshiba, Tokyo, Japan) using a commercially available solution (0.01 M citric acid monohydrate buffer pH 6.0; Sigma, St. Louis, MO) for a net boiling time of 20 min (MART-1) 26 and 35 min (CD3) 27 and cooled to room temperature. 27 Immunohistochemic staining using antihuman CD3 and Melan-A/MART-1 (Dako, Carpinteria, CA) was performed using peroxidase antiperoxidase procedure using 3-amino-9-ethylcarbazole (Dako) as the substrate chromogen. All sections were analysed by a single blind method and parameters determined on a 3 CCD image analysis system (DXC9 100P, Sony, Tokyo, Japan using KS400 software (Zeiss, Berlin, Germany).…”