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Objective To develop an antigen retrieval method for detection of human mammaglobin ( hMAM) immuno-histochemcal staining in old paraffin-embedded specimens .Methods The tissue sections in test group were put into dis-tilled water after deparaffinization and then moved into citric acid buffer ( pH 3.5) for 10-15 min.The other two meth-ods,microwave method and high pressure cooker method ,were compared as control groups at the same time .Finally, immu-nohistochemistry SP method was used to check the antibody in the sections .Results The color appearance in the test group (pH 3.5 citric solution) was better than that of microwave oven and high pressure cooker groups .In the test group, tissue sections were not easily cast off from the slices .Conclusion In this study,we have established a new and simple antigen retrieval method which will contribute to immunohistochemistry technology .
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The present study compared two heating methods currently used for antigen retrieval (AR) immunostaining: the microwave oven and the steam cooker. Myosin-V, a molecular motor involved in vesicle transport, was used as a neuronal marker in honeybee Apis mellifera brains fixed in formalin. Overall, the steam cooker showed the most satisfactory AR results. At 100 ºC, tissue morphology was maintained and revealed epitope recovery, while evaporation of the AR solution was markedly reduced; this is important for stabilizing the sodium citrate molarity of the AR buffer and reducing background effects. Standardization of heat-mediated AR of formalin-fixed and paraffin-embedded tissue sections results in more reliable immunostaining of the honeybee brain.
Subject(s)
Animals , Antigens/analysis , Bees/immunology , Immunohistochemistry/methods , Myosin Type V/analysis , Optic Lobe, Nonmammalian/chemistry , Antigens/immunology , Heating , Microwaves , Paraffin Embedding , Staining and LabelingABSTRACT
Objective To explore the effects of different methods of antigen retrieval on the results of immunohistochemical staining of nuclear receptors. Methods Antigens were retrieved by using microwave oven, autoclave, enzyme and autoclave plus enzyme, respectively, in liver sections from DA.1U rats, followed by immunohistochemical staining. Results After antigen retrieval with autoclave, the positive staining intensity of nuclear receptors LXR-α, LXR-β, PPAR-γ and FXR in the liver sections from DA.1U rats was enhanced obviously, and the location of nuclear receptors was better by using this method than the others. Conclusion In the immunohistochemical staining of nuclear receptors, using autoclave to retrieve antigens is the best method.
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Background Antigen retrieval method is the key of improving the successful rate of immunohistochemical assay in paraffin sections.To study an available method of antigen retrieval is a goal to achieve both good immunochemistry result and preserving retinal proteins.ObjectiveThe aim of present study is to investigate tyrosin digestion,high-temperature heat pressure,water bath heating and microwave repair in antigen retrieval for retinal tissues.MethodsRetinal tissue was isolated and obtained from clean Chinchilla rabbits.Four hundreds retinal paraffin sections were prepared.Four kinds of antigen retrieval methods for retinal tissue including tyrosin digestion,high-temperature heat pressure,water bath heating and microwave repair were used respectively.The depigmentated retinal paraffin section without antigen retrieval was used as control.The positive rates of expression of CRALBP,Rhodopsin and opsin proteins were evaluated and compared among four kinds of antigen retrieval methods by immunochemistry.ResultsCRALBP,Rhodopsin and opsin protein was positively expressed in cytoplasm of retinal pigment epithelial cells and the outer segment of photoreceptor respectively.No significant difference was found in the positive expression rates of these three proteins among four kinds of antigen retrieval methods (P＞0.05),but the differences in tissue integrity and background staining were statistically significant (P＜0.01).The structural damage of retina included loss and pucker of scalera,crack of nucleus and abnormal background stain in high-temperature heat pressure method,water bath heating method and microwave retrieval method.However,stable CRALBP,Rhodopsin and opsin protein expression and strain effectiveness,clear background without unspecific staining and integrated tissue were seen in tyrosin digestion method.ConclusionDuring the clinical pathology analysis of retinal tissue,the application of tyrosin digestion in antigen retrieval could obtain a better effectiveness.
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Objective To investigate the effect of antigen retrieval by microwave and the expression of CD 44 s and CD 44v6 in human intracranial metastastic tumor and human glioblastomas, and the relationship between the expression of CD 44 adhesion molecules and the invasion or metastasis of these tumors.Methods After antigen retrieval by microwave-0.1%Triton X-100(MW-T) and buffer solution of microwave-citrate salts(MW-CA), CD 44 s and CD 44v6 were used to detect the expression of CD 44 adhesion molecules in 20 normal human brain tissues, 35 human glioblastomas, and 30 intracranial metastastic tumor by microwave-LSAB immunohistochemical staining technique.Results Integral optical density(IOD) value of CD 44 s and CD 44v6 positive cells restored by MW-T and MW-CA was higher significantly than that of without antigen retrieval (P
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On the bases of the authors analysed 64 cases of pituitary adenoma, endocrinologic manifestations and immunohistochemical staining by both conventional means and the antigen retrieval system. The incidence of each type and there interrelationship was thus demonstrated. The endocrinologic results of 64 cases were as follows: Should the total number of each type of adenoma be included here. Prolactin-secreting adenoma(39.0%), nonfunctioning adenoma(26.6%), and GH-secreting adenoma(17.2%). Conventional immunohistochemical staining revealed the presence of null cell adenoma(48.4%), prolactinoma(21.9%), and GH-secreting adenoma(12.5%). All adenomas shown by conventional immunohistochemical staining to be null cell type were thus reclassified by the antigen retrieval system. On the basis of these results, the authors concluded that for the classification of pituitary adenomas, immunohistochemical staining by the antigen retrieval system is more sensitive than conventional immunohistochemical staining. In addition to avoid false-positive results, EM confirmation is required.
Subject(s)
Adenoma , Classification , Incidence , Lymphocytes, Null , Pituitary NeoplasmsABSTRACT
Objective To evaluate the influence of different antigen retrieval methods and pH values of fixatives on immunostaining of incised wound skin.Methods 3d-post-incision mouse skin were sampled and fixed in 4% formalin buffered with PBS at pH 5.0,6.0,7.0,8.0 and 9.0.The deparaffinized sections were treated 0.01mmol citrate buffuer(pH 6.0)in high pressure cooker or by microwave heating,or with pepsin,trypsin or microwave+pepsin.Thereafter,immunostainings of caspase-3,caspase-6,caspase-7,IL-8,and IL-10 were evaluated by immunohistochemical SABC method with ratio of immunostaining-positive cells,respectively.Results The immunostaining-positive cell ratios in sections treated by heating retrieval was higher than by proteinase treatment.The positive cell ratio was slightly higher by high pressure than by microwave heating.Sections were more easier peeled off during high pressure treatment than during micrwave heating.Sections treated with pepsin were fallen off easier than with trypsin digestion.The highest positive cell ratio was detected in sections by microwave+trypsin treatment without section peeling off among all antigen treatment methods.The best result was obtained in sections fixed in buffered formalin at pH 7.0 and 8.0.Conclusion The best immunostaining result may be obtained in mouse incised wound sections fixed in buffered formalin at pH 7.0 and 8.0 with microwave+trypsin treatment.