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S R Shi

Publications and source records attributed to S R Shi.

At least 19 recordsLinked to original sources
PubMed2001

Antigen retrieval immunohistochemistry and molecular morphology in the year 2001.

The impact of the antigen retrieval (AR) technique upon diagnostic immunohistochemistry (IHC) and upon research has been demonstrated by numerous of articles and more than a dozen major reviews. The specific aim of this survey of the field is to examine potential new approaches to retrieval of nucleic acid and protein from archival paraffin-embedded tissue for molecular biology-based diagnostic procedures that form the basis of the emerging field of molecular morphology. Any new approach must incorporate the principles of standardization and improved reproducibility. The ultimate goal will be to understand the mechanisms of fixation (by formalin) and "unfixation" (by AR). In the interim, the diligent application of practical procedures that have been shown to be tried and true is the least that we must demand from ourselves and our laboratories.

Antigens↗
PubMed2001

Antigen retrieval techniques: current perspectives.

Development of the antigen retrieval (AR) technique, a simple method of boiling archival paraffin-embedded tissue sections in water to enhance the signal of immunohistochemistry (IHC), was the fruit of pioneering efforts guided by the philosophy of rendering IHC applicable to routine formalin-fixed, paraffin-embedded tissues for wide application of IHC in research and clinical pathology. On the basis of thousands of articles and many reviews, a book has recently been published that summarizes basic principles for practice and further development of the AR technique. Major topics with respect to several critical issues, such as the definition, application, technical principles, and further studies of the AR technique, are highlighted in this article. In particular, a further application of the heat-induced retrieval approach for sufficient extraction of nucleic acids in addition to proteins, and standardization of routine IHC based on the AR technique in terms of a test battery approach, are also addressed. Furthermore, understanding the mechanism of the AR technique may shed light on facilitating the development of molecular morphology.

Animals↗
PubMed2001

Nitric oxide, prostanoids, cyclooxygenase, and angiogenesis in colon and breast cancer.

PURPOSE: Several studies have shown an overexpression of cyclooxygenase-2 (COX-2) and elevated levels of prostacyclin (PGI(2)) and thromboxane (TXA(2)) in colon cancer. In this report, we determined the distribution of inducible form of nitric oxide synthase (iNOS), PGI(2), and TXA(2) in cancerous and adjoining areas of specimens from human colon and breast cancer obtained during surgery. Additionally, we investigated differences in expression and histological localization of COX-2 in colon and breast cancer. EXPERIMENTAL DESIGN: Specimens were obtained during surgery, one centrally located, the second from an adjacent, cancer-free area. Activity of iNOS was determined, using the conversion of L-[(14)C]arginine to L-[(14)C]citrulline. PGI(2) and TXA(2) were measured as their stable metabolites, using enzyme immunoassay. A standard immunoperoxidase method was used for immunohistochemical expression of COX-2. RESULTS: Significant differences in iNOS, PGI(2), and TXA(2) expressions between colon and breast cancer were noted, with an enhanced expression of COX-2 in colon cancer, including the cancerous, adjoining, and stromatous fields. CONCLUSIONS: Increased expression of iNOS and production of prostanoids in colon cancer parallels the increase in COX-2, confirming the importance of this enzyme in colon cancer. The overexpression of COX-2, prostanoids, and nitric oxide in areas adjoining the tumor indicates increased metastatic potential for neoplastic cells in this area. Inflammatory changes in the tissue adjoining the cancer may play a role. COX-2 may result in the formation of new blood vessels and the spread of cancer.

6-Ketoprostaglandin F1 alpha↗
PubMed1999

Calcium-induced modification of protein conformation demonstrated by immunohistochemistry: What is the signal?

A recent study by Morgan et al. on the mechanism of the heating antigen retrieval (AR) has raised an interesting issue concerning calcium-induced modification of protein conformation demonstrated by immunohistochemistry (IHC). The current study is based on calcium-induced modification of thrombospondin (TSP) and Ki-67, as demonstrated by IHC using seven monoclonal antibodies (MAbs) to TSP and an MAb MIB1. Experiments were carried out on frozen tissue sections of bladder carcinoma and lymph node. Frozen sections were incubated with solutions of 50 mM CaCl2 and/or 10 mM EDTA at 4C overnight before formalin or acetone fixation for TSP and Ki-67, respectively. Sections were then fixed in 10% neutral buffered formalin or acetone before immunostaining. Seven MAbs to TSP, named Ab1 to 7 representing clone numbers of A4.1, D4.6, C6.7, A6.1, B5.2, A2.5, and HB8432, respectively, and MIB1 were utilized as primary antibodies. ABC was used as the detection system and AEC as the chromogen for immunohistochemical staining. An extracellular immunostaining pattern represented a positive result for TSP, and nuclear staining for MIB1. Frozen sections preincubated in 50 mM CaCl2 overnight at 4C showed significant loss of staining and/or altered staining pattern for six of the seven antibodies to TSP and MIB1 compared to positive controls not exposed to CaCl2. Lack of immunostaining of TSP and MIB1 attributable to exposure to CaCl2 could be partially recovered by incubating the frozen sections in EDTA. Calcium-induced modification of protein structure was demonstrated more than 10 years ago on the basis of immunochemical techniques. In this study, similar calcium-induced modification of protein was detectable by IHC in frozen tissue sections, suggesting that calcium-induced modification of protein structure may occur independently of fixation-induced modification. The fact that calcium binding may affect IHC staining is not surprising in view of the fact that antibody/antigen interactions are protein structure-dependent. However, in this experiment the change occurred before and independent of formalin fixation and does not necessarily imply a role for calcium in AR. There may be a valuable role for the use of chemical modification in visualization of protein structure changes in tissue sections by IHC. (J Histochem Cytochem 47:463-469, 1999)

Calcium↗
PubMed1998

Elevated and absent pRb expression is associated with bladder cancer progression and has cooperative effects with p53.

Rb protein (pRb) expression was evaluated in 185 cases of transitional cell carcinoma of the bladder from patients that underwent radical cystectomy. Tumors were stratified into three categories based on the percentage of nuclei expressing pRb: (a) 0, 0% of tumor cells showing nuclear reactivity; (b) 1+, 1-50% of tumor cells showing nuclear reactivity; and (c) 2+, >50% of tumor cells showing nuclear reactivity. Cases with undetectable (pRb 0) and high (pRb 2+) pRb reactivity had identical rates of recurrence. These cases had significantly higher recurrence (P = 0.0001) and lower survival rates (P = 0.0002) compared to cases with moderate (pRb 1+) pRb reactivity, indicating that high levels of pRb expression may reflect a dysfunctional (altered) Rb pathway. The tumors were also examined for alterations in p53 expression; patients with tumors altered in both p53 and pRb had significantly increased rates of recurrence (P < 0.0001) and survival (P < 0.0001) compared to patients with no alterations in either p53 or pRb; patients with alterations in only one of these proteins had intermediate rates of recurrence and survival. These results suggest that: (a) bladder cancers with high pRb expression do not show the tumor suppressor effects of the protein; and (b) alteration in both p53 and pRb may act in cooperative or synergistic ways to promote tumor progression.

Carcinoma, Transitional Cell↗
PubMed1998

Antigen retrieval immunohistochemistry used for routinely processed celloidin-embedded human temporal bone sections: standardization and development.

The use of immunohistochemistry (IHC) in routinely processed celloidin-embedded human temporal bone section has created a fruitful field in understanding the pathogenesis and pathophysiology of the human inner ear at a molecular level since the early 1990s when the antigen retrieval (AR) technique was developed. This review article focuses on several critical technical issues of AR technique based predominantly on our experiences and suggestions concerning further development and standardization of AR-IHC for IHC study of human temporal bone section, as well as other tissues embedded in celloidin. Examples of using AR-IHC in human temporal bone sections collected include our unpublished data in order to indicate the potential utility of this novel method. Suggestions of further development of AR techniques are proposed for references and comments.

Collodion↗
PubMed1998

MAP2 expression in developing dendrites of human brainstem auditory neurons.

Immunostaining of cytoskeletal elements has proved to be a useful technique for tracing ontogenetic development in the human central auditory system. In the present study, dendritic development in brainstem auditory nuclei (dorsal and ventral cochlear nuclei, medial and lateral superior olivary nuclei, and inferior colliculus) was studied using an antibody to a microtubule-associated protein, MAP2, a molecule which stabilizes dendritic processes by promoting assembly of microtubules. At 21-22 weeks of gestation, cells within the auditory nuclei first demonstrate cytoplasmic MAP2 immunoreactivity, but no dendritic structures have formed. Filamentous background staining at this stage may represent immunoreactivity in astrocytic processes. By the 24th fetal week, somata of auditory neurons are strongly immunostained and have developed short dendritic processes. During the perinatal period, dendrites extend up to 100-120 microm in length but are still sparsely branched and lack terminal formations. By the sixth postnatal month, neurons in all auditory nuclei have acquired dendritic arbors with a mature appearance. Thus MAP2 immunohistochemistry demonstrates that dendrogenesis in human brainstem auditory nuclei begins 16 weeks prior to term birth but does not reach the stage of mature dendritic morphology until several months into the postnatal period. This extended course of development implies a significant period of time during which neuronal activity could influence dendritic structure and function.

Auditory Pathways↗
PubMed1997

Axogenesis in the human fetal auditory system, demonstrated by neurofilament immunohistochemistry.

Morphological, electrophysiological and behavioral evidence indicates that the onset of rapid, synchronized conduction of auditory impulses occurs in the human brainstem during the 28th-29th fetal weeks. This implies that axonal connections in the brainstem auditory pathway are generated prior to this time. In order to investigate the sequence of axogenic events in the human brainstem pathway, we employed immunohistochemical techniques and an antibody to neurofilament protein. Immunostaining for axonal neurofilaments in an age-graded series of fetal brains demonstrates that a small number of cochlear nerve axons have invaded the ventral cochlear nucleus by the 16th fetal week. By this same time point, a limited number of trapezoid body-lateral lemniscus axons have reached the superior olivary complex and inferior colliculus. Between gestational weeks 16 and 26, there is marked expansion and collateralization of the ascending pathway from cochlear nerve to inferior colliculus. By week 26, ascending axons have begun to form plexuses of terminal neuropil within all of the brainstem auditory nuclei. Beginning in week 22, there is development of commissural axons (dorsal commissure of the lateral lemniscus and commissure of the inferior colliculus) and descending projections (descending collicular axons and olivocochlear bundle). This early establishment of a mature pattern of axonal connections presumably forms the basis for the appearance of myelin, acousticomotor reflexes and recordable brainstem responses by fetal week 29.

Auditory Pathways↗
PubMed1997

Antigen retrieval immunohistochemistry: past, present, and future.

The antigen retrieval (AR) technique, which is predominantly based on high-temperature heating of tissues, is used as a non-enzymatic pretreatment for immunohistochemical staining of formalin-fixed, paraffin-embedded tissue sections. It has been widely applied in pathology and analytical morphology. The existence of a growing body of literature on the AR technique raises a number of interesting issues for the further development of AR. These issues include the use of a "test battery" and the concept of "maximal retrieval" applied to the selection of optimal test protocols for the standardization of AR.

Animals↗
PubMed1996

Development of an optimal protocol for antigen retrieval: a 'test battery' approach exemplified with reference to the staining of retinoblastoma protein (pRB) in formalin-fixed paraffin sections.

The retinoblastoma (RB) gene, which encodes the nuclear RB protein (pRB), is believed to be involved in cell cycle control and cell differentiation. Studies have demonstrated that loss of RB function may play a role in tumour formation and progression of a variety of human tumours, such as bladder, lung, breast, and prostate cancers. The immunohistochemical detection of pRB expression in formalin-paraffin sections of human cancer has potential advantages of convenience, economy, and compatibility with routine surgical pathology practice. In practice, however, results using pRB antibodies on routinely processed, paraffin-embedded tissue have been inconsistent. In this study, the antigen retrieval (AR) method has been applied to the immunohistochemical detection of pRB in paraffin-embedded tissues and a 'test battery' approach has been developed to identify the principal variables that result in the optimal AR protocol. This approach includes the use of buffered solutions at pH 1, 6, and 10 with three different heating conditions (temperatures 120 degrees C, 100 degrees C, and 90 degrees C). In the example described here with antibody RB-WL-1, the low pH solution with the microwave heating at 100 degrees C proved most effective. Both fresh and routinely processed formalin-paraffin tissues of normal and bladder carcinoma were used for a comparison of the pRB immunostaining. The AR method was evaluated by comparing the immunohistochemical staining result on routinely processed formalin-paraffin sections with frozen sections of the same tumour. A consistent intensity of immunohistochemical staining for pRB was achieved using the identified optimal AR protocol on formalin-paraffin sections. All slides showed positive staining of pRB in normal mesenchymal and epithelial tissues. The pattern of pRB localization and intensity of staining was similar to that obtained in frozen sections, though the intensity obtained by AR treatment on paraffin sections was slightly to moderately stronger than that obtained in frozen sections. Once the protocol was identified, it was tested using routinely processed paraffin tissue sections of 245 cases of bladder carcinoma, with consistent pRB immunostaining results. The protocol described is simple to perform and gives reproducible results for evaluation of pRB expression by immunohistochemistry.

Cryopreservation↗
PubMed1996

Immunohistochemical detection of thrombospondin-1 in formalin-fixed, paraffin-embedded tissue.

Thrombospondin-1 (TSP) is a 450-KD glycoprotein that was initially discovered in the platelet alpha-granule. It now appears that TSP is intimately involved in the regulation of a variety of cellular functions and cell-to-cell interactions. Recently, it has been demonstrated that TSP functions as a p53-dependent inhibitor of angiogenesis in cultured fibroblasts from Li-Fraumeni patients and therefore may be an important factor involved with tumor invasion and metastasis. It has previously been demonstrated that TSP can be detected in frozen tissue sections by immunohistochemical methods. Our objective in this study was to determine the optimal antigen retrieval (AR) protocol for detection of TSP in formalin-fixed, paraffin-embedded tissue by using tissue sections from patients with invasive transitional cell carcinoma of the bladder. The optimal AR protocol was determined utilizing a variety of heating conditions and antigen retrieval buffers. Our results demonstrate that TSP can be reliably detected in paraffin-embedded tissue by immunohistochemical techniques that utilize AR with high-temperature microwave heating and a low-pH Tris-HCI buffer. The importance of this method is that it allows the reliable detection of TSP in archival tissue. This should facilitate further investigation into TSP's role in the regulation of cellular processes, including its influence on tumor angiogenesis and metastasis.

Carcinoma, Transitional Cell↗
PubMed1996

Use of pH 9.5 Tris-HCl buffer containing 5% urea for antigen retrieval immunohistochemistry.

Successful antigen retrieval (AR) immunohistochemistry is dependent on the temperature, heating time, and pH value of the AR solutions. There is no single standardized AR solution, however, that is suitable for all antibodies "routinely" used in surgical pathology for immunostaining archival tissue sections. We tested a variety of AR solutions varying in pH value, chemical composition, and molarity. Based upon preliminary results, we compared three AR solutions: 0.1 M Tris-HCl buffer, pH 9.5, containing 5% urea, 0.1 M Tris-HCl buffer pH 9.5 without urea, and citrate buffer, pH 6.0. Each AR solution was tested with a panel of 34 antibodies using microwave heating for antigen retrieval. The heating conditions were standardized at 10 min and an automated stainer was used to standardize the immunostaining method. The Tris-HCl containing urea was superior to pH 6.0 citrate buffer for 22 antibodies. In 12 cases, Tris-HCl with urea was also superior to Tris-HCl alone. In 12 cases, the intensity was similar for all three retrieval solutions. The staining obtained with Tris-HCl with urea was equal to or better than with pH 6.0 citrate buffer in all cases. The Tris-HCl with urea solution is satisfactory for AR of most antibodies employed in routine surgical pathology.

Antigens↗
PubMed1996

Comparative study of antigen retrieval heating methods: microwave, microwave and pressure cooker, autoclave, and steamer.

We present a study comparing the most popular heating methods currently used for antigen retrieval (AR) immunostaining: the microwave oven, microwave with pressure cooker, autoclave, and steamer heating. A panel of 21 antibodies was tested on formalin fixed, paraffin embedded sections using these heating methods and Tris-HC1 buffer, pH 9.5, plus 5% urea as the AR solution. Three observers independently evaluated the intensity of AR immunostaining. All heating methods yielded good results for AR immunostaining. There were only minor differences among the heating methods for AR when the optimal concentration of primary antibody for normal immunostaining was used; however, background staining may occasionally be troublesome if antibodies are not retitrated and diluted further for use on tissues after AR. Significant differences were observed only after further dilution of the primary antibodies: the microwave pressure cooker, extended microwave heating (5 min x 4) and autoclave heating then showed a similar intensity of staining that was stronger than results obtained with the steamer (20 min) or regular microwave heating (5 min x 2). Extension of the steamer heating time, however, yielded equivalent results. This study indicates that different heating methods can yield similar intensities of AR immunostaining if the heating times are adjusted appropriately. It is noteworthy that, in general, the adjusted conditions for maximal retrieval differ from those most widely cited in the literature, or recommended by manufacturers. That several heating devices may provide similar results permits the use of different AR heating methods according to the equipment available. This study also is an early step in standardizing the AR immunostaining protocol by providing uniform conditions for "maximal retrieval" as a common end point for all laboratories.

Antigens↗
PubMed1995

Detection of occult bone marrow micrometastases in patients with operable lung carcinoma.

OBJECTIVES: A large proportion of patients with operable lung carcinoma (no evidence of systemic spread of tumor) develop metastatic disease after primary therapy. More sensitive and specific methods are needed to identify patients at highest risk for recurrence who may benefit most from adjuvant therapy, while sparing those patients who do not require such treatment. SUMMARY BACKGROUND DATA: Using epithelial-specific monoclonal antibodies, the authors have developed an immunocytochemical assay capable of detecting as few as 2 lung cancer cells in 1 million bone marrow cells. METHODS: The assay was used to test the bone marrow (from resected ribs) of 43 patients with primary non-small cell lung carcinoma who showed no clinical or pathologic evidence of systemic disease. RESULTS: Occult bone marrow micrometastases (BMMs) were detected in 40% of patients (17/43) with non-small cell lung cancer, including 29% (5/17) of patients with stage I or II disease and 46% of whom (12/26) had stage III disease. The median follow-up was 13.6 months. Patients with occult BMMs had significantly shorter times to disease recurrence compared with patients without BMMs (7.3 vs. > 35.1 months, p = 0.0009). Furthermore, for patients with stage I or II disease, the presence of occult BMMs was significantly associated with a higher rate of recurrence (p = 0.0004). CONCLUSIONS: The detection of occult BMMs identifies patients with operable non-small cell lung carcinoma who are at significantly increased risk for recurrence, independent of tumor stage, and may be useful in evaluating patients for adjuvant treatment protocols.

Antibodies, Monoclonal↗
PubMed1995

Antigen retrieval immunohistochemistry under the influence of pH using monoclonal antibodies.

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.

Antibodies, Monoclonal↗