Paraffin tissue sections microwave treatment

Igarashi H, Sugimura H, Maruyama K, et al. Alteration of immunoreactivity by hydrated autoclaving, microwave treatment, and simple heating of paraffin-embedded tissue sections. APMIS 1994 102 295-307. 

Ki-67 antigen immunohistochemical staining is a simple and reliable procedure for studying tumor proliferative activity in frozen or formalin-fixed, paraffin-embedded tissues, including archival specimens. This antigen can be retrieved on sections of formalin-fixed and paraffin-embedded tissues by autoclave treatment (Fig. 10.1) or microwave heating (Fig. 10.2). Both methods are reliable and are presented later. 

Henke, R.-P., and Ayhan, N. 1994. Enhancement of hybridization efficiency in interphase cytogenetics on paraffin-embedded tissue sections by microwave treatment. Analyt. Cell. Pathol. 6 319-325. 

Igarashi, H., Sugimura, H., Maruyama, K., Kitayama, Y., Ohta, I., Suzuki, M., Tanaka, M., Dobashi, Y., and Kino, I. 1994. Alteration of immunoreactivity by hydrated autoclaving, microwave treatment, and simple heating of paraffin-embedded tissue sections. APMIS 102 295-307. 

Despite some successes with the above pretreatments, the development of wet heat-induced epitope retrieval (HIER) procedures, which involves heating the fixed tissue sections in dilute metal-salt or buffer solutions at or above 100°C, for several minutes to 1/2 h, was the critical breakthrough in paraffin section immunohistochemistry (2, 7-9). Today, there are many variations of the original HIER technique. These differ primarily in the recommended buffer solutions and/or the source or mode of heating, but the basic formula of wet heat treatment over a fixed time period is similar. The most popular HIER technologies use microwave ovens, stainless steel or plastic pressure cookers, autoclaves, vegetable steamers or water-baths as the heat sources and low molarity buffers with acidic or alkaline pH (8,9,11-14). 

Biopsy tissue specimens are fixed with formalin and embedded in paraffin. Sections (5 xm thick) are mounted on silanated slides, heated at 56°C for 1 hr, deparaffinized in xylene, rehydrated in graded ethanols, and rinsed in distilled water. They are placed in 0.01 M citrate buffer (pH 6.0) and heated in a microwave oven for six cycles of 5 min each, followed by cooling at room temperature for 20 min. Endogenous peroxidase activity is blocked with hydrogen peroxide in distilled water for 8 min, and nonspecific background staining is prevented by treatment with nonimmune horse serum for 20 min. 

Gastric tumor tissue is fixed with 4% neutral formaldehyde for 1 day and embedded in paraffin (Kitayama et al., 2000). Paraffin sections (6 xm thick) are deparaffinized with xylene and rehydrated with ethanol. Centromeric a-satellite DNA probes and locus-specific identifier probes (c-myc and p53) are available from Vyis Inc. (Downers Grove, IL). The probes are labeled with orange (Cy 3) or green (PITC) using digoxigenin-ll-dUTP and nick translation. The sections are placed in 0.01 M citrate buffer (pH 6.0) and heated in a microwave oven for 10 min. This is followed by treatment with 0.2% pepsin in 0.01N HC1 for 10 min at 37°C, and then exposure to 0.1% NP40/2 X SSC for 10 min at the same temperature. 

Kasper et al. (1994) reported the selective im-munohistological localisation of protein disulphide isomerase in human type II alveolar and bronchial epithelial cells. The detection of the hidden antigen with the monoclonal antibody 5B5 usually failed in paraffin sections but succeeded after microwave pre-treatment of tissue slices. 

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