Immunoperoxidase Staining Of Paraffin Sections Research Articles

A euthymic hairless mouse model of Helicobacter pylori colonization and adherence to gastric epithelial cells in vivo.

The hairless mouse strain NS:Hr/ICR was examined as a potential small animal model of Helicobacter pylori colonization, adherence to gastric epithelial cells in vivo, and gastritis. Among several small animals tested, NS:Hr/ICR mice proved to be the most highly susceptible to H. pylori infection. Challenge with clinical isolates of H. pylori consisting of either phenotype I or II (VacA and CagA positive and negative, respectively) resulted in colonization by mucus-resident and epithelial cell-adherent bacterial populations. Cell-adherent bacteria resisted 80 cycles of top-speed Vortex washing and were recovered only by homogenization of serially washed glandular stomach tissue, indicating intimate association with the mucosal surface. Immunoperoxidase staining of paraffin sections of gastric tissue from infected mice revealed H. pylori antigens localized in the glandular region of the mucosa, with some colonized areas seen in the vicinity of submucosal mononuclear cell infiltration. The latter inflammatory reaction was observed as a function of the H. pylori phenotype (only type I induced inflammation) and the challenge dose (only those mice challenged with 10(8) CFU or higher showed the reaction). The NS:Hr/ICR strain of mice is a suitable miniature model of H. pylori infection and may prove useful in the quest for an efficacious mode of treatment for this common infection in humans.

Identification of bipotential progenitor cells in human liver development

Intermediate filament proteins have been reported to be expressed in a cell lineage-specific manner during morphogenesis. We studied the expression of cytokeratin (CK)14, CK19, and vimentin and of the hepatocyte-specific HepPar1 antigen during the development of human liver. Nineteen fetal livers (gestational ages 4 to 40 weeks), 3 normal infant livers, and 3 normal adult livers were studied by immunoperoxidase staining of paraffin sections with monoclonal anti-CK19, anti-vimentin, and HepPar1 antibodies and polyclonal anti-CK14 antibodies. Double-immunostaining for CK14 and CK19 as well as bile duct cytokeratin and HepPar1 antigen was also done. CK19 and HepPar1 antigen were the first markers detected in immature progenitor cells of the liver primordium at 4 weeks' gestation. During subsequent liver development, the progenitor cells expressed HepPar1 antigen, CK14, and CK19, from 8 to 14 weeks' gestation. As hepatocyte differentiation progressed, expression of HepPar1 antigen increased, and CK14 and CK19 were abrogated from hepatoblasts at 14 to 16 weeks' gestation. In contrast, as progenitor cells transformed into ductal plate cells, CK19 expression increased and persisted in differentiated bile ducts, whereas CK14 and HepPar1 antigen were lost. Vimentin was detected in ductal plate and biliary epithelial cells from 9 to 36 weeks' gestation, but not in hepatoblasts or hepatocytes. Double-immunostaining confirmed coexpression of CK14 and CK19 in the progenitor cells for a short time (8 to 14 weeks' gestation) during early development. Double immunostaining for bile duct CK and HepPar1 antigen clearly demonstrated the divergence of the hepatocyte and bile duct epithelial cell lineages. Our findings suggest that hepatic progenitor cells differentiate in steps marked by the acquisition or loss of specific phenotypic characteristics. Commitment of the HepPar1+CK19+ progenitor cells to either hepatocyte or bile duct epithelial cell lineages results in increased expression of one marker and loss of the other marker. These characteristics clearly identify bipotential hepatic progenitor cells in the developing human liver.

Identification of bipotential progenitor cells in human liver development

Intermediate filament proteins have been reported to be expressed in a cell lineage-specific manner during morphogenesis. We studied the expression of cytokeratin (CK)14, CK19, and vimentin and of the hepatocyte-specific HepPar1 antigen during the development of human liver. Nineteen fetal livers (gestational ages 4 to 40 weeks), 3 normal infant livers, and 3 normal adult livers were studied by immunoperoxidase staining of paraffin sections with monoclonal anti-CK19, anti-vimentin, and HepPar1 antibodies and polyclonal anti-CK14 antibodies. Double-immunostaining for CK14 and CK19 as well as bile duct cytokeratin and HepPar1 antigen was also done. CK19 and HepPar1 antigen were the first markers detected in immature progenitor cells of the liver primordium at 4 weeks' gestation. During subsequent liver development, the progenitor cells expressed HepPar1 antigen, CK14, and CK19, from 8 to 14 weeks' gestation. As hepatocyte differentiation progressed, expression of HepPar1 antigen increased, and CK14 and CK19 were abrogated from hepatoblasts at 14 to 16 weeks' gestation. In contrast, as progenitor cells transformed into ductal plate cells, CK19 expression increased and persisted in differentiated bile ducts, whereas CK14 and HepPar1 antigen were lost. Vimentin was detected in ductal plate and biliary epithelial cells from 9 to 36 weeks' gestation, but not in hepatoblasts or hepatocytes. Double-immunostaining confirmed coexpression of CK14 and CK19 in the progenitor cells for a short time (8 to 14 weeks' gestation) during early development. Double immunostaining for bile duct CK and HepPar1 antigen clearly demonstrated the divergence of the hepatocyte and bile duct epithelial cell lineages. Our findings suggest that hepatic progenitor cells differentiate in steps marked by the acquisition or loss of specific phenotypic characteristics. Commitment of the HepPar1+CK19+ progenitor cells to either hepatocyte or bile duct epithelial cell lineages results in increased expression of one marker and loss of the other marker. These characteristics clearly identify bipotential hepatic progenitor cells in the developing human liver. (Hepatology 1996 Mar;23(3):476-81)

Identification of bipotential progenitor cells in human liver development

Intermediate filament proteins have been reported to be expressed in a cell lineage-specific manner during morphogenesis. We studied the expression of cytokeratin (CK)14, CK19, and vimentin and of the hepatocyte-specific HepPar1 antigen during the development of human liver. Nineteen fetal livers (gestational ages 4 to 40 weeks), 3 normal infant livers, and 3 normal adult livers were studied by immunoperoxidase staining of paraffin sections with monoclonal anti-CK19, anti-vimentin, and HepPar1 antibodies and polyclonal anti-CK14 antibodies. Double-immunostaining for CK14 and CK19 as well as bile duct cytokeratin and HepPar1 antigen was also done. CK19 and HepPar1 antigen were the first markers detected in immature progenitor cells of the liver primordium at 4 weeks' gestation. During subsequent liver development, the progenitor cells expressed HepPar1 antigen, CK14, and CK19, from 8 to 14 weeks' gestation. As hepatocyte differentiation progressed, expression of HepPar1 antigen increased, and CK14 and CK19 were abrogated from hepatoblasts at 14 to 16 weeks' gestation. In contrast, as progenitor cells transformed into ductal plate cells, CK19 expression increased and persisted in differentiated bile ducts, whereas CK14 and HepPar1 antigen were lost. Vimentin was detected in ductal plate and biliary epithelial cells from 9 to 36 weeks' gestation, but not in hepatoblasts or hepatocytes. Double-immunostaining confirmed coexpression of CK14 and CK19 in the progenitor cells for a short time (8 to 14 weeks' gestation) during early development. Double immunostaining for bile duct CK and HepPar1 antigen clearly demonstrated the divergence of the hepatocyte and bile duct epithelial cell lineages. Our findings suggest that hepatic progenitor cells differentiate in steps marked by the acquisition or loss of specific phenotypic characteristics. Commitment of the HepPar1+CK19+ progenitor cells to either hepatocyte or bile duct epithelial cell lineages results in increased expression of one marker and loss of the other marker. These characteristics clearly identify bipotential hepatic progenitor cells in the developing human liver.

Proliferating cell nuclear antigen in prostatic adenocarcinoma: Correlation with established prognostic indicators

Objective. The utility of an antibody to proliferating cell nuclear antigen (PCNA), a growth-specific nuclear protein, was assessed as a prognostic variable for prostatic adenocarcinoma. Its expression was correlated with established prognostic indicators, including tumor grade, stage, prostattc-specific antigen (PSA), and percent of tumor in the gland at excision. Methods. Forty archival needle biopsies containing a minimum of four hundred tumor cells were analyzed. Immunoperoxidase staining of paraffin sections was performed for PCNA (PC 10) after pretreatment in antigen retrieval solution. A proliferative index (PI) for each case was derived using image analysis with measurement of at least four hundred twenty-five nuclei. Results. PI values ranged from 2.4 to 31.3 percent. Mean PI values varied significantly (ANOVA, p = 0.005) among cases with dominant Gleason grade (DGG) of 3 (mean PI = 9.3%), 4 (mean PI = 13.7%), and 5 (mean PI = 18.8%). By t test, significant differences were noted for PI in cases with DGG 2 and 3 versus those with DGG 4 and 5 ( p = 0.0065). PI for cases with DGG 3 versus 5 showed significant difference (p = 0.0017). Tumors of Gleason scores 5 to 7 differed significantly from those with scores 8 to 10 (p = 0.014). A statistical relationship for PI and PSA, clinical stage, and percent tumor at resection could not be established by linear regression. Conclusions. These findings suggest that additional study of the PI, as determined by PCNA immunohistochemistry and image analysis, may be warranted to determine its usefulness as an adjunctive parameter in prostate adenocarcinoma. This technique may be particularly useful in needle biopsies where limited tumor may render assessment of grade difficult.

Non‐Hodgkin's lymphoma of the major salivary gland: a morphologic and immunohistochemical study of 15 cases

A total of 15 cases of Japanese salivary gland lymphoma was analysed by conventional histology and also by immunoperoxidase staining of paraffin sections. The initial sites included: parotid gland (14 cases, 93%) and submandibular gland (1 case, 7%). The mean age was 59 yr, with a male to female ratio of 2.8:1. The only one patient (Case 9) with clinical evidence of Sjögren's syndrome showed the development of intrasalivary malignant lymphoma. Each lymphoma was classified according to the Working Formulation for Clinical Usage. The Working Formulation grades were 20% low, 67% intermediate, and 13% high. These neoplasms fell into six categories: follicular, small cleaved type (1 case); follicular, mixed small cleaved and large cell type (2 cases); follicular, large cell type (2 cases); diffuse, mixed small and large cell type (1 case); diffuse, large cell type (7 cases); and large cell, immunoblastic type (2 cases). Fifteen tumors were studied using a indirect immunoperoxidase method. A selected antibody panel was used, including B-cell markers (immunoglobulins, Ki-B3, L26), T-cell markers (UCHL-1 and MT1) and histiocytic markers (lysozyme, alpha 1-antitrypsin and cathepsin D). Eleven cases reacted with pan-B-marker (Ki-B3 and L26) and eight of them showed the presence of monoclonal cytoplasmic immunoglobulin (C-Ig). Following rigid criteria, monoclonal C-Ig of M kappa was demonstrated in one case of follicular, small cleaved cell type, one follicular, large cell type and two diffuse, large cell type; M lambda was demonstrated in one diffuse, large cell type; and G lambda was demonstrated in one follicular, mixed small cleaved and large cell type, and two diffuse, large cell type.(ABSTRACT TRUNCATED AT 250 WORDS)

Accelerated Immunogold Silver and Immunoperoxidase Staining of Paraffin Sections with the Use of Microwave Irradiation: Factors Influencing Results

With the use of microwave irradiation, incubation times for the immunotechniques used in immunomicroscopy can be shortened significantly. The authors found that factors influencing results are irradiation time, power output, and presence of gold label. Precise temperature control is essential, and techniques to achieve this are discussed. For beta-human chorionic gonadotropin (beta-HCG), the results with the immunoperoxidase technique were inferior to those of the conventional technique, and with the immunogold silver technique an enhancement of labeling results was achieved. Immunogold silver staining for beta-HCG on paraffin sections can be performed with the use of microwave irradiation within one hour.

Eradication of herpes simplex virus persistence in rat trigeminal ganglia by retrograde axoplasmic transport

Potential use of retrograde axoplasmic flow to eradicate virus persistence in ganglionic cells was studied in a new herpes simplex virus (HSV) persistence model in rat trigeminal ganglia. After injection of the F strain of HSV type 1 into the mental nerve, viral antigens were detectable in the ganglia by the immunofluorescence and peroxidase methods between postinoculation (p.i.) days 3 and 6 but not thereafter. None of 82 inoculated rats showed signs of acute illness, and some survived for more than 502 days without symptoms. By cocultivation of ganglion tissues with Vero cells, the virus was isolated from 42 of 49 ganglia (85.7%) between 15 and 386 days (p.i.). HSV DNA was solely localized in the nucleus of neurons by immunoperoxidase staining of paraffin sections with a biotinylated HSV DNA probe, and the presence of HSV DNA-positive cells was confirmed in four of four ganglia on p.i. day 6 and in five of six on p.i. day 502. The efficacy of axoplasmic flow in drug delivery to ganglionic cells was investigated by injection of doxorubicin (ADM) into the nerve once used for virus inoculation. As early as 19 h after injection, strong ADM-specific autofluorescence was seen in the nuclei of neurons parental to the mental nerve and in those of adjacent Schwann cells, and the death of ADM-positive cells subsequently ensued. A single injection of ADM reduced the virus isolation rate from 31/37 (84%) to 3/37 (8%).

Lymphoid Tumors of the Ocular Adnexa: Clinical Correlation With the Working Formulation Classification and Immunoperoxidase Staining of Paraffin Sections

Eighty-eight patients with ocular adnexal lymphoid lesions examined at the Mayo Clinic between 1970 and 1982 were evaluated. The histopathologic type was reclassified using the working formulation. Paraffin sections were evaluated with immunoperoxidase staining. There were 11 “pseudotumors” and 77 lymphomas. Sixty lymphomas were small lymphocytic type A; 27 tissues had a monoclonal staining pattern, and 4 had a polyclonal staining pattern. Five of the pseudotumors had a polyclonal staining pattern; none had a monoclonal pattern. The remainder did not stain or tissue was not available. Thirty-six of the 44 lymphomas that presented in the ocular adnexa remained localized. There were no distinguishing features between conjunctival and orbital lymphomas. Significant damage to the ocular tissue occurred in 15 of the 63 patients treated with radiation therapy. Older, asymptomatic patients with a favorable histologic type may not require treatment; therapy is reserved for specific indications. The probability of survival to five or seven years differs little from that expected in the general population.

The use of monoclonal antibodies to estrogen receptors (ER) for immunoperoxidase detection of ER in paraffin sections of human breast cancer tissue.

Two monoclonal antibodies against MCF-7 human estrogen receptors were used for immunoperoxidase staining of paraffin sections of human breast cancer tissue. The staining was predominantly located in the nucleus of epithelial cells. Variation in the staining intensity was observed among individual cells. A significant positive correlation between the number of positively stained cells and cytosol estrogen receptor content (fmol of bound estrogen/mg of protein) was observed. The potential and the limitations of the present techniques are discussed.

Simian virus 40-associated fatal interstitial pneumonia and renal tubular necrosis in a rhesus monkey.

Simian virus 40 was isolated from a young adult rhesus monkey dying of naturally acquired interstitial pneumonia and renal tubular necrosis. Light microscopic examination demonstrated basophilic intranuclear inclusions associated with the lesions in the lung and the kidney. These inclusions were positive for antigen of simian virus 40 by immunoperoxidase staining of paraffin sections of affected organs. Electron microscopy revealed characteristic papovarirus particles in these tissues. This study is the first report indicating that simian virus 40 may produce pulmonary and renal disease in its natural host.

Detection of viral proteins in mouse mammary tumors by immunoperoxidase staining of paraffin sections.

An indirect immunoperoxidase method is described, which can readily detect viral antigens in paraffin sections of primary, transplanted, and metastatic mammary tumors of mice. In addition to having the obvious advantage of not being limited to fresh specimens, immunoperoxidase staining of paraffin sections proved to be superior in many respects when compared with immunofluorescence and frozen sections. Immunoperoxidase staining of paraffin sections is permanent and provides the kind of histological detail required for precise cytological identification and localization with light microscopy. All of 25 tumors and 4 metastatic lesions showed evidence of glycoprotein gp52 as well as other mouse mammary tumor viral antigens. The pattern and intensity of the stain were related to the degree of histologic differentiation of the tumor. Wide variations in expression of viral antigens by individual malignant cells were observed within the same tumor.