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Antigen retrieval (AR) incorporating high-temperature microwave (MW) heating of tissue sections before immunostaining is a revolutionary technique that can unmask the antigens in formalin-fixed tissue sections, thus making them available for immunohistochemical staining. Although high temperature is believed to be the primary mechanism in retrieval of antigens, a variety of chemical solutions have been tested to define an optimal AR solution. We tested the hypothesis that pH of the AR solution may influence the quality of immunostaining by using seven different AR buffer solutions at a series of different pH values ranging from 1 to 10. We evaluated the staining of monoclonal antibodies to cytoplasmic antigens (AE1, HMB45, NSE), nuclear antigens (MIB-1, PCNA, ER), and cell surface antigens (MT1, L26, EMA) on routinely formalin-fixed, paraffin-embedded sections under different pH conditions with MW heating for 10 min. The intensity of immunostaining was graded in a blinded fashion. The pH value of the AR buffer solution was carefully measured before, immediately after, and 15 min after the AR procedure. The influence of pH on AR immunohistochemical staining can be summarized into three patterns. Some antigens (L26, PCNA, AE1, EMA, and NSE) showed excellent retrieval throughout the pH range. Other antigens (MIB1 and ER) showed strong intensity of immunohistochemical staining at very low pH and at neutral to high pH, but a dramatic decrease in the intensity of the AR immunostaining at moderately acidic pH (pH 3-6). Still others (MT1 and HMB45) showed increasing intensity of the AR immunostaining with increasing pH, but only weak immunostaining at low pH. Among the seven buffer solutions at any given pH value, the intensity of AR immunostaining was very similar. However, Tris-HCl buffer tended to produce better results at higher pH, compared with other buffers. Although high-temperature heating is believed to be the most important factor for the AR technique, the pH value of the AR solution is an important co-factor for some antigens. Optimization of the AR system should therefore include optimization of the pH of the AR solution. Our results indicate that AR immunostaining of Tris-HCl or sodium acetate buffer at pH 8-9 may be suitable for most antigens, although certain nuclear antigens show optimal staining at low pH.

The adipocyte-rich microenvironment forms a niche for ovarian cancer metastasis, but the mechanisms driving this process are incompletely understood. Here we show that salt-inducible kinase 2 (SIK2) is overexpressed in adipocyte-rich metastatic deposits compared with ovarian primary lesions. Overexpression of SIK2 in ovarian cancer cells promotes abdominal metastasis while SIK2 depletion prevents metastasis in vivo. Importantly, adipocytes induce calcium-dependent activation and autophosphorylation of SIK2. Activated SIK2 plays a dual role in augmenting AMPK-induced phosphorylation of acetyl-CoA carboxylase and in activating the PI3K/AKT pathway through p85α-S154 phosphorylation. These findings identify SIK2 at the apex of the adipocyte-induced signaling cascades in cancer cells and make a compelling case for targeting SIK2 for therapy in ovarian cancer.

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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)

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