Isoxanthohumol improves obesity and glucose metabolism via inhibiting intestinal lipid absorption with a bloom of Akkermansia muciniphila in mice.

OBJECTIVE: Polyphenols have health-promoting effects, such as improving insulin resistance. Isoxanthohumol (IX), a prenylated flavonoid found in beer hops, has been suggested to reduce obesity and insulin resistance; however, the mechanism remains unknown. METHODS: High-fat diet-fed mice were administered IX. We analyzed glucose metabolism, gene expression profiles and histology of liver, epididymal adipose tissue and colon. Lipase activity, fecal lipid profiles and plasma metabolomic analysis were assessed. Fecal 16s rRNA sequencing was obtained and selected bacterial species were used for in vitro studies. Fecal microbiota transplantation and monocolonization were conducted to antibiotic-treated or germ-free (GF) mice. RESULTS: The administration of IX lowered weight gain, decreased steatohepatitis and improved glucose metabolism. Mechanistically, IX inhibited pancreatic lipase activity and lipid absorption by decreasing the expression of the fatty acid transporter CD36 in the small intestine, which was confirmed by increased lipid excretion in feces. IX administration increased markers of intestinal barrier function, including thickening the mucin layer and increasing caludin-1, a tight-junction related protein in the colon. In contrast, the effects of IX were nullified by antibiotics. As revealed using 16S rRNA sequencing, the microbial community structure changed with a significant increase in the abundance of Akkermansia muciniphila in the IX-treated group. An anaerobic chamber study showed that IX selectively promoted the growth of A. muciniphila while exhibiting antimicrobial activity against some Bacteroides and Clostridium species. To further explore the direct effect of A. muciniphila on lipid and glucose metabolism, we monocolonized either A. muciniphila or Bacteroides thetaiotaomicron to GF mice. A. muciniphila monocolonization decreased CD36 expression in the jejunum and improved glucose metabolism, with decreased levels of multiple classes of fatty acids determined using plasma metabolomic analysis. CONCLUSIONS: Our study demonstrated that IX prevents obesity and enhances glucose metabolism by inhibiting dietary fat absorption. This mechanism is linked to suppressing pancreatic lipase activity and shifts in microbial composition, notably an increase in A. muciniphila. These highlight new treatment strategies for preventing metabolic syndrome by boosting the gut microbiota with food components.

The potential diagnostic significance of crypt differentiation in gastric dysplasia.

AIMS: This study investigated the relationship between the differentiation of tumour cells into crypts, which is determined by cell differentiation into Paneth and neuroendocrine cells, and tumour infiltration in gastric dysplasia. METHODS AND RESULTS: The lesions were endoscopically biopsied low-grade dysplasia (LGD), endoscopically resected high-grade dysplasia (HGD) or cancer with submucosal invasion. LGD (n = 32) displayed crypt differentiation across the entire width of the tumour in all cases. Crypt differentiation was identified as a characteristic of tumours with low biological malignancy. HGD (n = 40) included tumours with a mixture of areas with and without crypt differentiation (n = 25) and tumours with crypt differentiation throughout the entire width (n = 15). Of the cancers with submucosal invasion (n = 30), the morphological progression of the HGD area with crypt differentiation, the HGD area without crypt differentiation and invasive cancer without crypt differentiation was confirmed for 23 samples. In two lesions, invasive cancer without crypt differentiation developed from HGD without crypt differentiation throughout the tumour width. In five samples, well-differentiated tubular adenocarcinoma with crypt differentiation developed from HGD with crypt differentiation and invaded with lamina propria-like stroma. CONCLUSIONS: Loss of crypt differentiation could be an objective indicator of infiltration in the progression of HGD to invasive cancer. The invasive potential of dysplasia depends upon the presence or absence of crypt differentiation.

Interleukin-32 regulates downstream molecules and promotes the invasion of pancreatic cancer cells.

Pancreatic cancer is a malignant neoplasm with high invasiveness and poor prognosis. In a previous study, a highly invasive pancreatic cancer cell line was established and found to feature enhanced interleukin-32 (IL-32) expression. However, whether IL-32 promotes the invasiveness by enhancing or suppressing the expression of IL-32 through regulating downstream molecules was unclear. To investigate the effect of IL-32, cells were established with high levels of expression or downregulated IL-32; their invasive ability was measured using a real-time measurement system and the expression of some candidate downstream molecules involved in invasion was evaluated in the two cell types. The morphological changes in both cell types and the localization of IL-32 expression in pancreatic cancer tissues were studied using immunohistochemistry. Among the several splice variants of IL-32, cells transfected with the ε isoform had increased invasiveness, whereas the IL-32-suppressed cells had reduced invasiveness. Several downstream molecules, whose expression was changed in the two cell types, were monitored. Notably, changes of E-cadherin, CLDN1, CD44, CTGF and Wnt were documented. The morphologies of the two cell types differed from the original cell line. Immunohistochemically, the expression of IL-32 was observed only in tumor cells and not in normal pancreatic cells. In conclusion, IL-32 was found to promote the invasiveness of pancreatic cancer cells by regulating downstream molecules.

Utility of Ethylene-Diamine-Tetraacetic Acid Buffer Solution With Boric Acid for Immunostaining of Specimens Stored for an Extended Period.

Antigen modification and denaturation are recognized causes of false negatives in immunostaining. Specimens that have been stored for an extended period at room temperature show decreased immunoreactivity and may mislead the diagnosis. Studies of the molecular targeting of drugs often involve immunostaining of previous samples and, in some situations, only unstained specimens can be used. The present study aimed to develop an effective staining method to recover antigen activation in unstained specimens stored for an extended period by using ethylene-diamine-tetraacetic acid (EDTA) buffer solution with boric acid. We compared several commonly used antigen retrieval solutions and found that Tris-borate-EDTA (TBE) buffer solution with a pH ≥8.3 provided sufficient antigen retrieval. However, pH values higher than 8.3 (9.0, 10.0, and 11.0) frequently caused severe tissue damage. Thus, TBE with pH 8.3 was the most suitable antigen retrieval solution for recovering the antigenicity of specimens stored for an extended period. This procedure may allow useful immunohistochemical information, even from sections that have been stored for an extended period.

CD206+ macrophage is an accelerator of endometriotic-like lesion via promoting angiogenesis in the endometriosis mouse model.

In endometriosis, M2 MΦs are dominant in endometriotic lesions, but the actual role of M2 MΦ is unclear. CD206 positive (+) MΦ is classified in one of M2 type MΦs and are known to produce cytokines and chemokines. In the present study, we used CD206 diphtheria toxin receptor mice, which enable to deplete CD206+ cells with diphtheria toxin (DT) in an endometriosis mouse model. The depletion of CD206+ MΦ decreased the total weight of endometriotic-like lesions significantly (p < 0.05). In the endometriotic-like lesions in the DT group, a lower proliferation of endometriotic cells and the decrease of angiogenesis were observed. In the lesions, the mRNA levels of VEGFA and TGFß1, angiogenic factors, in the DT group significantly decreased to approximately 50% and 30% of control, respectively. Immunohistochemical study revealed the expressions of VEGFA and an endothelial cell marker CD31 in lesions of the DT group, were dim compared to those in control. Also, the number of TGFß1 expressing MΦ was significantly reduced compared to control. These data suggest that CD206+ MΦ promotes the formation of endometriotic-like lesions by inducing angiogenesis around the lesions.

Melatonin sensitises shikonin-induced cancer cell death mediated by oxidative stress via inhibition of the SIRT3/SOD2-AKT pathway.

Recent research suggests that melatonin (Mel), an endogenous hormone and natural supplement, possesses anti-proliferative effects and can sensitise cells to anti-cancer therapies. Although shikonin (SHK) also possesses potential anti-cancer properties, the poor solubility and severe systemic toxicity of this compound hinders its clinical usage. In this study, we combined Mel and SHK, a potentially promising chemotherapeutic drug combination, with the aim of reducing the toxicity of SHK and enhancing the overall anti-cancer effects. We demonstrate for the first time that Mel potentiates the cytotoxic effects of SHK on cancer cells by inducing oxidative stress via inhibition of the SIRT3/SOD2-AKT pathway. Particularly, Mel-SHK treatment induced oxidative stress, increased mitochondrial calcium accumulation and reduced the mitochondrial membrane potential in various cancer cells, leading to apoptosis. This drug combination also promoted endoplasmic reticulum (ER) stress, leading to AKT dephosphorylation. In HeLa cells, Mel-SHK treatment reduced SIRT3/SOD2 expression and SOD2 activity, while SIRT3 overexpression dramatically reduced Mel-SHK-induced oxidative stress, ER stress, mitochondrial dysfunction and apoptosis. Hence, we propose the combination of Mel and SHK as a novel candidate chemotherapeutic regimen that targets the SIRT3/SOD2-AKT pathway in cancer.

Establishment of highly invasive pancreatic cancer cell lines and the expression of IL-32.

Compared to tumors of other organs, pancreatic cancer is highly aggressive; with one of its biological features being that, despite a prominent fibrotic stroma, there is remarkable infiltration of tumor cells. This characteristic is considered to be the main reason for the poor prognosis of patients with pancreatic cancer. Therefore, in order to elucidate the factors that contribute to this high invasiveness, a selective invasion method was used to establish four highly invasive subclones from six human pancreatic cancer cell lines. The results demonstrated that two cell lines did not exhibit enhanced invasiveness. Microarray analysis revealed that, in the highly invasive cell lines, several genes were expressed at high levels, compared with the original cell lines. These highly expressed genes were recognized only in highly invasive cells. Among them, IL-32 was most strongly upregulated in the highly invasive cells, compared with cells with a low invasive potential, as well as the original cells. RT-qPCR and western blot analysis confirmed the high levels of expression of IL-32 in highly invasive cells at the RNA and protein levels. In addition, immunohistochemical analysis of resected surgical materials revealed that the tumor cells expressed IL-32 and, in particular, many IL-32 positive cells were seen at the invasive front of the tumor tissue. IL-32 is a cytokine that is widely involved in the development of cancer and has recently received considerable attention. This cytokine has multiple splice variants and shows a wide variety of behaviors, depending on the tumor type and primary organ. Although some hypotheses have been proposed to explain the activity of IL-32, a unified view has not been agreed. In the present study, through the establishment of highly invasive cells from pancreatic cancer and a comprehensive gene analysis, it is suggested that IL-32 may serve an important role as a molecule involved in the invasiveness of this neoplasm.

CD11c+ M1-like macrophages (MΦs) but not CD206+ M2-like MΦ are involved in folliculogenesis in mice ovary.

Macrophages (MΦs) are involved in folliculogenesis and ovulation. However, it is unknown which type of MΦ, M1 or M2, plays a more essential role in the ovary. CD206 or CD11c diphtheria toxin receptor transgenic (DTR) mice, which enable depletion of CD206+ M2 MΦs and CD11c+ MΦ or CD11c+ Dendritic cells (DCs), respectively, were used. Oocytes were used for in vitro fertilization and embryo transfer. In vitro fertilized embryos derived from M2 MΦ depleted oocytes were transferred to pseudo pregnant wild type mice. CD11c DTR mice were also used to investigate the role of CD11c cells, M1 MΦ and DCs in folliculogenesis. In WT mice, the proportion of CD206+ M2-like MΦs was not increased in follicular induction, while that of CD11c+ M1-like MΦs was increased. In CD206 DTR mice, folliculogenesis was normal and the ovulation number, fertilization rate, and implantation rate were similar to those in WT mice. In CD11c DTR mice, folliculogenesis was impaired with ovarian hemorrhage and the staining of platelet derived growth factor-receptor ß (PDGF-Rß), a marker of pericytes, and CD34, a marker of endothelial cells, was reduced. CD11c+ cells, M1 MΦs or DCs, may be involved in folliculogenesis, while M2 MΦs are not involved in folliculogenesis.

Endoglin (CD105) and SMAD4 regulate spheroid formation and the suppression of the invasive ability of human pancreatic cancer cells.

In this study, we investigated the ability of pancreatic cancer cell lines to form spheroids with the aim of identifying factors involved in cell invasiveness, a property that leads to a poor prognosis in pancreatic cancer. For this purpose, 8 cell lines derived from human pancreatic cancer tissues were cultured in non-adherent culture conditions to form spheroids, as well as normal monolayers. The morphology of the cells was observed and spheroid diameters measured. mRNA expression was compared between cells cultured under both culture conditions. The gene knockdown of endoglin (ENG) and SMAD4, components of the transforming growth factor-ß (TGF-ß) signaling system, using siRNAs was conducted in spheroids in order to identify affected protein signaling factors, determine the morphological changes occurring over time and to measure the invasive capacity of the cells constituting spheroids. The cell lines exhibited differences in their spheroid-forming abilities. The expression of SMAD4 and ENG concomitantly increased in the cells that formed spheroids. SMAD4 was transported into the nucleus when spheroids were formed. The expression of ENG was decreased in the cells in which SMAD4 was knocked down; by contrast, the expression of BMP and activin membrane-bound inhibitor (BAMBI) and noggin (NOG), further components of the TGF-ß signaling system, increased. In the cells in which ENG was knocked down, the decreased mRNA expression of TGF-ß receptor type 2 (TGFBR2) and SMAD9 was observed, as well as a change in the expression of pSMAD1/5/9, and a tendency of spheroids to decrease in size. Spheroids cultured on Matrigel exhibited a tendency towards a reduction in size over time, as well as a tendency to invade into the Matrigel. In particular, the cells in which ENG was knocked down exhibited spheroids which were reduced in size, and also exhibited an increase in invasiveness, and a decrease in adhesiveness. Thus, our data indicate that in pancreatic cancer cells, the expression of ENG may be controlled by a pathway mediated by SMAD4. In addition, ENG was found to be related to the spheroid-forming ability of cells and to be involved in the invasive capacity of pancreatic cancer cells.

Facilitatory effect of insulin treatment on hepatocellular carcinoma development in diabetes.

BACKGROUND: To evaluate the effect of insulin treatment on the incidence and/or severity of hepatocellular carcinoma (HCC) in a mouse model of HCC based on diabetes. METHODS: We recently reported that neonatal streptozotocin (STZ) treatment causes type 1 diabetes and subsequent HCC in ddY, Institute for Animal Reproduction (DIAR) mice. Newborn male DIAR mice were divided into three groups based on STZ and insulin (INS) treatment. STZ was subcutaneously injected (60 mg/g) into the STZ-treated group (DIAR-nSTZ mice, N = 13) and the STZ/insulin-treated group (DIAR-nSTZ/INS mice, N = 20). A physiologic solution was injected into the control group (DIAR-control mice, N = 8) 1.5 days after birth. Insulin was subcutaneously injected into the DIAR-nSTZ/INS mice according to the following protocol: 2 IU/day at 4-5 weeks of age, 3 IU/day at 5-7 weeks of age, and 4 IU/day at 7-12 weeks of age. All mice were fed a normal diet and were subjected to physiological and histopathological assessments at 12 weeks of age. RESULTS: DIAR-nSTZ mice had significantly lower body weight and higher blood glucose levels than DIAR-control mice, whereas no significant differences were observed between DIAR-nSTZ/INS mice and control mice. At 12 weeks of age, lower weight of paratesticular fat and higher levels of total cholesterol, triglyceride, and free fatty acids were observed in DIAR-nSTZ mice compared to DIAR-control mice, whereas there were no significant differences between DIAR-nSTZ/INS mice and DIAR-control mice. In the livers of DIAR-nSTZ mice, HCC was observed in 15% of cases, and dysplastic nodules were observed in 77% of cases. In the livers of DIAR-nSTZ/INS mice, HCC was observed in 39% of cases and dysplastic nodules were observed in 61% of cases (p = 0.011). Moreover, the average tumor size was significantly larger in STZ/INS-treated mice than in STZ-treated mice. Immunohistochemical analysis demonstrated that the expression of ERK1/2, downstream substrates of insulin signaling that activate cell proliferation, was significantly higher in STZ/INS-treated mice compared to STZ-treated mice. CONCLUSIONS: Insulin treatment promoted, rather than inhibited, the progression of liver carcinogenesis in DIAR-nSTZ mice. Hyperinsulinemia rather than hyperglycemia can accelerate the progression of HCC via insulin signaling.

Aberrant iron metabolism might have an impact on progression of diseases in Tsumura Suzuki obese diabetes mice, a model of spontaneous metabolic syndrome.

Tsumura Suzuki obese diabetes (TSOD) mice spontaneously develop obesity and type 2 diabetes with aberrant accumulation of excessive iron in the spleen. Aberrantly accumulated iron may cause oxidative stress and result in various symptoms of metabolic syndrome in the mice. We investigated iron metabolism and oxidative stress in TSOD mice. Male TSOD and control mice were killed at 2, 3, 6, and 8 months of age, and blood and tissue samples were collected. The serum levels of ferritin and oxidized low-density lipoprotein (OxLDL) were measured. Total glutathione concentrations of liver and spleen were also measured. Serum ferritin and OxLDL were higher in TSOD mice than in control mice at 2 and 6 months. In addition, the glutathione concentrations in TSOD mice were lower in the liver and higher in the spleen at 3 and 6 months than those in control mice. These results suggest that abnormal iron metabolism and imbalanced oxidative stress occurs in young and old TSOD mice. We propose herein that TSOD mice might be a unique and valuable model for investigating the role of iron metabolism in pathogenesis of metabolic syndrome.

Histopathological characteristics of glutamine synthetase-positive hepatic tumor lesions in a mouse model of spontaneous metabolic syndrome (TSOD mouse).

We previously reported that Tsumura-Suzuki obese diabetic (TSOD) mice, a polygenic model of spontaneous type 2 diabetes, is a valuable model of hepatic carcinogenesis via non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). One of the characteristics of tumors in these mice is the diffuse expression of glutamine synthetase (GS), which is a diagnostic marker for hepatocellular carcinoma (HCC). In this study, we performed detailed histopathological examinations and found that GS expression was diffusely positive in >70% of the hepatic tumors from 15-month-old male TSOD mice. Translocation of ß-catenin into nuclei with enhanced membranous expression also occurred in GS-positive tumors. Small lesions (<1 mm) in GS-positive cases exhibited dysplastic nodules, with severe nuclear atypia, whereas large lesions (>3 mm) bore the characteristics of human HCC, exhibiting nuclear and structural atypia with invasive growth. By contrast, the majority of GS-negative tumors were hepatocellular adenomas with advanced fatty change and low nuclear grade. In GS-negative tumors, loss of liver fatty acid-binding protein expression was observed. These results suggest that the histological characteristics of GS-positive hepatic tumors in TSOD mice resemble human HCC; thus, this model may be a useful tool in translational research targeting the NAFLD/NASH-HCC sequence.

Complete Resolution of Pseudomalignant Erosion in a Reflux Gastroesophageal Polyp with Proton Pump Inhibitor.

Pseudomalignant erosion is a diagnostic pitfall for pathologists in the differential diagnosis of malignant neoplasms. Here, we present a challenging case of a biopsy specimen from the eroded head of a polyp at the esophagogastric junction. A malignant neoplasm could not be ruled out due to the presence of bizarre stromal cells. A second biopsy performed after the administration of a proton pump inhibitor (PPI) for 4 weeks revealed endoscopic resolution of the polyp along with the complete histological resolution of the bizarre stromal cells and led to the diagnosis of pseudomalignant erosion in a reflux gastroesophageal polyp. In conclusion, histological and endoscopic response to PPI therapy is an important clue for the correct diagnosis of reflux gastroesophageal polyps with pseudomalignant erosion.

Bone metastasis in gastrointestinal stromal tumors preferentially occurs in patients with original tumors in sites other than the stomach.

Bone metastases are rare in gastrointestinal stromal tumors (GISTs) and data on the clinicopathological profiles are lacking. The purpose of this report was to identify the clinicopathological profiles of this rare clinical setting by evaluating 23 cases, four of which were our own and the additional 19 were from the relevant English literature. In 18 cases, the primary GISTs occurred in sites other than the stomach, although a high proportion of these tumors do arise in the stomach. All tumors at the disease presentation had more than a low risk of recurrence, with most tumors either at a high risk or initially malignant with liver metastasis. In four cases, bone metastasis was the primary metastatic manifestation. Although rare in GISTs, bone metastasis should be considered in patients with primary tumors at a high risk for recurrence or in initially malignant tumors with liver metastasis, especially with primary tumors in sites other than the stomach.

Neonatal streptozotocin treatment causes type 1 diabetes and subsequent hepatocellular carcinoma in DIAR mice fed a normal diet.

PURPOSE: Hepatocellular carcinoma (HCC) represents a major health challenge because of its increasing morbidity and mortality. The establishment of useful models of HCC can significantly contribute to unveiling its pathophysiology. We developed a novel mouse model of HCC based on type 1 diabetes and reported its histopathological features. METHODS: Newborn male ddY, Institute for Animal Reproduction (DIAR) mice were divided into two groups on the basis of streptozotocin treatment, which induces type 1 diabetes. Streptozotocin was subcutaneously injected (60 mg/g) into the treated group (DIAR-nSTZ mice), whereas physiologic solution was injected into the control group (DIAR-control mice) at 1.5 days after birth. All mice were fed a normal diet and histopathologically assessed at 6, 8, 10, 12, 19, and 27 weeks of age. RESULTS: At 8 weeks, small hepatocytic nodules with mild to moderate cellular atypia were observed in the livers of DIAR-nSTZ mice, which progressed to large hepatocytic nodules with cellular atypia and infiltrating growth at 12 weeks, identical to those in well-differentiated human HCC. At 19 and 27 weeks, moderately differentiated HCC was observed in all DIAR-nSTZ mice. Conversely, no neoplastic findings were evident in DIAR-control mice. No steatosis or fibrosis was evident in either group. Immunohistochemical analysis revealed that all nodules observed in DIAR-nSTZ mice were positive for glutamine synthetase. CONCLUSIONS: In DIAR-nSTZ mice, the development of HCC with similarity to human HCC and high reproducibility can be achieved using a short and simple protocol. We believe that this model will be useful for studying liver carcinogenesis.

Development of an ultrasound-emitting device for performing rapid immunostaining procedures.

Although intraoperative rapid diagnosis is conventionally performed using hematoxylin-eosin (HE)-stained specimens, the use of additional special staining, together with immunostaining techniques, has been examined in recent years to improve diagnostic accuracy. In intraoperative rapid diagnosis, immunostaining should be completed within 7-10 min, because the pathologist is typically presented with an HE-stained specimen within the same time period. We hypothesized that ultrasound may enhance antigen-antibody reactions and reduce the number of immunostaining steps. To clarify the ability of ultrasound to support immunostaining, we first created an ultrasonic generator specifically for immunostaining. Next, we explored the optimal conditions for immunostaining of formalin-fixed specimens to examine the utility of the ultrasonic generator. Finally, we tried immunostaining with the ultrasonic generator using frozen specimens to simulate intraoperative rapid diagnosis. We report herein that ultrasound enables immunostaining of frozen specimens in approximately 10 min.

Freshly prepared immune complexes with intermittent microwave irradiation result in rapid and high-quality immunostaining.

We have previously reported that intermittent microwave irradiation shortened the primary or secondary antibody incubation time to 10 min in a special moist chamber. To achieve precise immunostaining within 1h, we attempted to generate a novel procedure, "freshly prepared immune complex with intermittent microwave irradiation (f-IC-M)". The advantage of this immunostaining procedure lies in a one-step incubation instead of primary and then secondary antibody application. In this study, we employed five primary antibodies to examine the efficiency and quality of this procedure. As expected, every primary antibody examined brought about precise immunostaining within 45 min for formalin-fixed, paraffin-embedded sections, and within 15 min for frozen sections. In addition, this procedure is able to generate double-immunoenzymatic staining with different enzyme-labeled primary antibodies if desired. As any combination of primary and secondary antibodies is possible by this one-step application, f-IC-M increases the efficiency of immunostaining without losing quality. Therefore, this procedure is able to rapidly provide diagnostic information to the pathologists.

Improved 1-h rapid immunostaining method using intermittent microwave irradiation: practicability based on 5 years application in Toyama Medical and Pharmaceutical University Hospital.

Immunostaining depending on antigen-antibody specificity is the commonest approach for determining the localization of specific antigens in tissue sections. This procedure is applicable not only with frozen or specially fixed samples, but also has proved reliable with formalin-fixed paraffin-embedded tissue sections through improvement of antigen-retrieval. Immunostaining is thus firmly established as a tool for diagnostic pathology and in our institute multiple antibodies are applied for 13-15% of the cases examined, as well as H and E staining. With the standard approach, approximately 3 h is necessary from the beginning of deparaffinization till covering sections with the Envision system. We utilized intermittent microwave irradiation for 10 min during hybridization with primary and secondary antibodies in a special moist-chamber, to achieve all immunostaining steps within 1 h in 178 primary antibodies frequently used for diagnostic pathology. According to our 5 years experience, such microwave irradiation not only obtained significant specific staining for enhancing the specificity of antigen-antibody reactions, but also inhibited nonspecific binding. We present herein the details of the methodology and recommendations for its application with particular primary antibodies. This method can contribute to savings in time and energy, allowing pathologists to rapidly obtain diagnostic information.