scholarly journals Validation of the S-phase specificity of histone (H3) in situ hybridization in normal and malignant cells.

1996 ◽  
Vol 44 (3) ◽  
pp. 221-226 ◽  
Author(s):  
A M Gown ◽  
J J Jiang ◽  
H Matles ◽  
M Skelly ◽  
T Goodpaster ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Mrna Expression ◽  
Tritiated Thymidine ◽  
S Phase ◽  
Phase Cell ◽  
Cell Type ◽  
Ki 67 ◽  
Double Labeling ◽  
Proliferation Indices

Several different methods of measuring proliferation indices have been developed, including measurements of cellular DNA content (flow cytometry), S-phase incorporation of thymidine analogues into DNA (e.g., tritiated thymidine and 5'-bromodeoxyuridine), and immunostaining of cell cycle-restricted proteins (e.g., Ki-67 antigen and PCNA). Theoretical and practical problems with each method have made it difficult to compare absolute proliferation rates among cells of different lineages and degrees of malignancy. More recently, in situ hybridization (ISH) for histone 3 (H3) mRNA has been introduced. We used a double labeling method for comparing H3 mRNA expression and S-phase incorporation of 5'-bromodeoxyuridine (BrdU) to determine if H3 mRNA expression was tightly associated with S-phase in a variety of malignant and nontransformed cell types. In addition, labeling results were compared in methacarn- and formalin-fixed tissues to extend the potential usefulness of H3 ISH, using a postfixation technique for the alcohol-fixed specimens. As expected for a cumulative marker, variation was noted in the percentage of the BrdU-positive cells double labeled with H3 ISH (53-89%), depending on cell type and length of BrdU incubation. In contrast, the percentage of the H3 ISH-positive cell population double labeled for BrdU was independent of the cell type of BrdU incubation time (mean 78%). Similarly, a consistent percentage of H3 ISH-positive cell populations was double labeled for BrdU in normal tissues (mean 97%). These findings support a well-conserved timing mechanism for H3 mRNA expression and DNA replication. We conclude that H3 ISH is an extremely accurate technique for assessment of S-phase cell proliferation indices.

The localization of laminin mRNA and protein in the postimplantation embryo and placenta of the mouse: an in situ hybridization and immunocytochemical study

Development ◽  
1988 ◽  
Vol 104 (3) ◽  
pp. 431-446 ◽  
Author(s):  
P.V. Senior ◽  
D.R. Critchley ◽  
F. Beck ◽  
R.A. Walker ◽  
J.M. Varley
Keyword(s):  
In Situ Hybridization ◽  
Connective Tissue ◽  
Basement Membrane ◽  
Mrna Expression ◽  
Lamina Propria ◽  
Muscularis Mucosa ◽  
Cell Type ◽  
Parietal Endoderm ◽  
Muscularis Externa

In situ hybridization (ISH) and immunocytochemistry were used to localize sites of synthesis and deposition of the basement membrane glycoprotein laminin during development in the postimplantation mouse embryo and extraembryonic membranes. In addition, similar studies were performed on postnatal viscera during the first 20 days after birth. Up to 10 days post coitum, embryonic laminin synthesis was confined to parietal endoderm. In maternal tissue, intense laminin mRNA expression was detected in decidual cells in the mesometrial and antimesometrial endometrium at 5–7 days. At 10 days, uniform expression was still seen within the mesometrial endometrium, with higher levels around migrating trophoblast, but in the antimesometrial aspect expression was restricted to the basal zone. High levels of mRNA expression persisted in parietal endoderm throughout gestation but much lower levels were detected in visceral yolk sac. In the mature placenta, laminin mRNA expression was also found associated with fetal vessels in the labyrinth and giant cells at the fetal/maternal boundary. In the embryo, the external limiting membrane of the cerebral vesicles and spinal cord stained for laminin protein and detectable mRNA was found in the pia mater. Growing peripheral nerves and dorsal and ventral root fibres expressed laminin mRNA and stained for laminin protein. Laminin mRNA expression was found in ureteric buds and nephrogenic vesicles (but not in metanephric blastema) during early prenatal kidney development, and in glomeruli, Bowman's capsule, loops of Henle and collecting duct cells at later stages of development, and after birth. All these structures possessed laminin-rich basement membrane (BM). Laminin mRNA expression fell to below detectable levels in the kidney around weaning. In the gut, laminin expression and protein staining was confined to the muscularis externa and the lamina propria during embryogenesis. After birth, the muscularis externa, muscularis mucosa and lamina propria cells corresponding to fibroblasts had detectable laminin mRNA, but in adult gut no laminin mRNA could be demonstrated in any cell type. In liver, low levels of laminin mRNA were seen in the capsule and in periportal connective tissue. After birth, laminin mRNA was associated with intrahepatic bile channels; no laminin mRNA was detected in the parenchyma and protein deposition was restricted to blood sinus BM. In the adult liver, no laminin mRNA was detected in any cell type. The developing heart showed uniform expression of laminin mRNA from 12 days to before birth. Postnatally, labelling was restricted to connective tissue cells.

scholarly journals Double labeling with non-isotopic in situ hybridization and BrdU immunohistochemistry: calmodulin (CaM) mRNA expression in post-mitotic neurons of the olfactory system.

1992 ◽  
Vol 40 (4) ◽  
pp. 535-540 ◽  
Author(s):  
S Biffo ◽  
L Verdun di Cantogno ◽  
A Fasolo
Keyword(s):  
In Situ Hybridization ◽  
Mrna Expression ◽  
Simultaneous Detection ◽  
Brdu Incorporation ◽  
Birth Date ◽  
Pregnant Rats ◽  
Double Labeling ◽  
Olfactory Neuroepithelium ◽  
Brdu Immunohistochemistry

We describe a novel histochemical procedure for simultaneous detection of mRNA expression by in situ hybridization (ISH) and DNA synthesis on cells that are pulse-labeled with bromodeoxyuridine (BrdU) by immunohistochemistry (ICC). Pregnant rats were injected with BrdU at embryonic Day 20 and the olfactory bulbs of their pups were collected daily. The expression of calmodulin (CaM) mRNA was analyzed by ISH with an anti-sense digoxigenin-labeled riboprobe and BrdU incorporation by indirect ICC. Starting 5 days after BrdU injection, a few tufted and granular neurons of the olfactory bulb were observed to be double labeled for CaM mRNA and BrdU. To study the olfactory neuroepithelium, adult animals were injected with BrdU, sacrificed after 30 days, and the nasal mucosa dissected and decalcified. The co-expression of CaM mRNA and BrdU incorporation was then analyzed in the olfactory neuroepithelium: BrdU-positive primary olfactory neurons were also CaM mRNA positive. The combination of ISH and ICC on the same section resulted in improved BrdU staining with respect to both increased intensity and reduced background levels. The procedure described here can be applied to a variety of problems in developmental biology and is of potential value for correlating the timing of specific mRNA expression with the birth date of a cell type of interest.

scholarly journals Determination of Hormone Receptors, Human Epidermal Growth Factor Receptor 2 and Ki67 Status in Invasive Breast Carcinoma: A Concordance Study between Immunohistochemistry, Fluorescence in Situ Hybridization, and GeneXpert® Breast Cancer STRAT4 Assay

2021 ◽  
Author(s):  
rajaa elaje ◽  
Abdellah Naya ◽  
Ayoub KHOAJA ◽  
Younes Zaid ◽  
Mounia Oudghiri ◽  
...  
Keyword(s):  
Breast Cancer ◽  
In Situ Hybridization ◽  
Mrna Expression ◽  
Hormone Receptors ◽  
Therapy Response ◽  
Cancer Prognosis ◽  
University Hospital ◽  
Automated System ◽  
Ki 67

Abstract BackgroundThe accurate assessment of hormone receptors (ER and PR), HER2 and Ki-67 proliferative index provides meaningful information about breast cancer prognosis and prediction of therapy response. Immunohistochemistry, the most common method for evaluating these prognostic biomarkers, can be impacted by numerous variabilities due to pre-analytical/analytical factors and subjective interpretation by pathologists. The Xpert® Breast Cancer STRAT4, a RT-qPCR based system, can be used to classify breast invasive carcinomas based on the assessment of these 4 biomarkers. In this study, we investigated the accuracy of RT-qPCR based mRNA expression levels in a closed, single-use cartridge, automated system compared with the current gold standard, immunohistochemistry (IHC), and fluorescent in situ hybridization (FISH) for HER2 equivocal cases.Methods We evaluated ESR1, PGR, ERBB2 and MKi67 mRNA expression by Xpert Breast Cancer STRAT4 and ER, PR, HER2 and Ki67 by IHC (FISH for HER2 IHC 2+) in 200 formalin-fixed paraffin-embedded (FFPE) tissue blocks with invasive breast cancer, collected from the Pathology Department of Casablanca Ibn Rochd University Hospital.Results Concordance between Xpert ® Breast Cancer STRAT4 and IHC was 93.5% for ER, 83.51% for PR, 95% for HER2 (92% for IHC+FISH), and 81.20% for Ki67 (excluding intermediate IHC Staining 10 ≤ %IHC <20). The simple Kappa coefficient was, for ER, 0.830 (P < 0, 0001), 0.565 (P < 0, 0001) for PR, 0.838 (P < 0, 0001) for HER2-IHC, 0.771 (P< 0, 0001) for HER2 IHC+FISH and, for, Ki67, 0.458 (P < 0, 0001).Conclusions We demonstrated globally a high concordance between centrally assessed IHC, IHC+FISH and mRNA measurements of ER/ESR1 and HER2/ERBB2, and a moderate agreement between PR/PGR and Ki67/MKi67. These findings provide an additional, objective, and quantitative assessment of tumor receptor status in breast cancer.

Expression of transforming growth factor alpha during development

1988 ◽  
Vol 66 (8) ◽  
pp. 1113-1121 ◽  
Author(s):  
V. K. M. Han ◽  
A. J. D'Ercole ◽  
D. C. Lee
Keyword(s):  
In Situ Hybridization ◽  
Growth Factor ◽  
Mrna Expression ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Alpha ◽  
Egf Receptors ◽  
Hybridization Histochemistry ◽  
In Situ Hybridization Histochemistry ◽  
Factor Alpha

Transforming growth factors (TGFs) are polypeptides that are produced by transformed and tumour cells, and that can confer phenotypic properties associated with transformation on normal cells in culture. One of these growth-regulating molecules, transforming growth factor alpha (TGF-α), is a 50 amino acid polypeptide that is related to epidermal growth factor (EGF) and binds to the EGF receptor. Previous studies have shown that TGF-α is expressed during rodent embryogenesis between 7 and 14 days gestation. To investigate the cellular sites of TGF-α mRNA expression during development, we have performed Northern analyses and in situ hybridization histochemistry on the conceptus and maternal tissues at various gestational ages. Contrary to previous reports, both Northern analyses and in situ hybridization histochemistry indicate that TGF-α mRNA is predominantly expressed in the maternal decidua and not in the embryo. Decidual expression is induced following implantation, peaks at day 8, and declines through day 15 when the decidua is being resorbed. In situ hybridization revealed that expression of TGF-α mRNA is highest in the region of decidua adjacent to the embryo and is low or nondetectable in the uterus, placenta, and embryo. In addition, we could not detect TGF-α mRNA expression in other maternal tissues, indicating that the induction of TGF-α transcripts in the decidua is tissue specific, and not a pleiotropic response to changes in hormonal milieu that occur during pregnancy. The developmentally regulated expression of TGF-α mRNA in the decidua, together with the presence of EGF receptors in this tissue, suggests that this peptide may stimulate mitosis and angiogenesis locally by an autocrine mechanism. Because EGF receptors are also present in the embryo and placenta, TGF-α may act on these tissues by a paracrine or endocrine mechanism.

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