human breast carcinoma tissue sections  (WuXi AppTec)

Journal: Nucleic Acids Research

Article Title: Spatially multiplexed RNA in situ hybridization to reveal tumor heterogeneity

doi: 10.1093/nar/gkz1151

Figure Lengend Snippet: Local RNA in situ hybridization. ( A ) Schematic overview of a microfluidic implementation of chromogenic RNA in situ hybridization. The initial preparation of the tissue section including paraffin removal (1), heat (2) and protease (3) pretreatment, as well as fixation was performed globally. The primary probe was then hybridized locally to the RNA of interest (4). In this case the detection of HER2 is shown with internal negative ( dapB ) and positive ( ActB ) controls. Finally, the amplification of the signal using preamplifier and amplifier strands (5), the binding of enzyme (6), and the enzymatic reactions (7) were performed globally on the whole slide. ( B ) Fluorescence images of RNA-ISH experiments detecting the gene expression of HER2 in cell pellet sections of the breast cancer cell lines MCF7 (left), SKBR3 (center) and BT474 (right). Probes directed against bacterial dapB and ActB were used as negative and positive controls, respectively. Scale bar: 100 μm.

Article Snippet: Breast carcinoma tissue sections were either obtained from BioChain (T2235086) or were kindly provided and anonymized by the tissue biobank of the Department of Pathology and Molecular Pathology of the Universitätsspital Zürich.

Techniques: RNA In Situ Hybridization, Amplification, Binding Assay, Fluorescence, Expressing

Journal: Nucleic Acids Research

Article Title: Spatially multiplexed RNA in situ hybridization to reveal tumor heterogeneity

doi: 10.1093/nar/gkz1151

Figure Lengend Snippet: Detection of HER2 expression status by RNA in situ hybridization using internal positive and negative controls. ( A ) Fluorescence images of the signals detected for HER2 , dapB , and ActB in a mammary carcinoma section. Scale bar: 100 μm. ( B ) Quantitative analysis of the fluorescence intensity of FISH signals detected for HER2 , dapB , and ActB integrated per sectioned cell for FFPE sections of the breast cancer cell lines MCF7, SKBR3, and BT474 (see Figure ) as well as the mammary carcinoma from (A). 100 ms excitation.

Article Snippet: Breast carcinoma tissue sections were either obtained from BioChain (T2235086) or were kindly provided and anonymized by the tissue biobank of the Department of Pathology and Molecular Pathology of the Universitätsspital Zürich.

Techniques: Expressing, RNA In Situ Hybridization, Fluorescence

Journal: Nucleic Acids Research

Article Title: Spatially multiplexed RNA in situ hybridization to reveal tumor heterogeneity

doi: 10.1093/nar/gkz1151

Figure Lengend Snippet: Single color multiplexed RNA-ISH for the detection of breast cancer biomarkers. ( A ) The primary probes for ER , PgR , and HER2 were delivered to spatially distinct regions of a mammary carcinoma section using a microfluidic chip (schematic in upper left corner). Fluorescence images of the signal detected for the breast cancer biomarkers ER , PgR and HER2 in a mammary carcinoma section. Scale bar: 100 μm. ( B ) Fluorescence intensity of FISH signals detected for ER , PgR and HER2 integrated per sectioned cell for FFPE sections of the breast cancer cell lines MCF7, SKBR3 and BT474 (see ) as well as the mammary carcinoma from (A). 500 ms excitation.

Article Snippet: Breast carcinoma tissue sections were either obtained from BioChain (T2235086) or were kindly provided and anonymized by the tissue biobank of the Department of Pathology and Molecular Pathology of the Universitätsspital Zürich.

Techniques: Fluorescence

Journal: Nucleic Acids Research

Article Title: Spatially multiplexed RNA in situ hybridization to reveal tumor heterogeneity

doi: 10.1093/nar/gkz1151

Figure Lengend Snippet: Spatially multiplexed RNA-ISH to detect spatial tumor heterogeneity in mammary carcinoma sections. ( A ) Fluorescence images of RNA-ISH signals detected for HER2 and ActB by global ISH or local ISH experiments on mammary carcinoma sections. Nuclei visualized using DAPI. The images display the inter- and intratumor heterogeneity of the HER2 expression. Scale bar: 100 μm. ( B , C ) Integrated fluorescence intensity of the HER2 signal per sectioned cell in three different regions of a strongly (B) (35 HER2 copies, HER2 IHC 3+) and low (C) (five HER2 copies, HER2 IHC 2+) HER2 -expressing carcinoma tissue section (left). Using a Mann–Whitney test, the Cohen effect size ( r ) for interregional heterogeneity was calculated from the combined areas (I–IV) within a region and displayed by the values given in the graph on the left. A matrix of Cohen effect sizes calculated for the individual areas within or between regions (intraregional and interregional heterogeneity) is shown on the right. Corresponding regions are shown on hematoxylin and immunohistochemically stained tissue sections in .

Article Snippet: Breast carcinoma tissue sections were either obtained from BioChain (T2235086) or were kindly provided and anonymized by the tissue biobank of the Department of Pathology and Molecular Pathology of the Universitätsspital Zürich.

Techniques: Fluorescence, Expressing, MANN-WHITNEY, Staining

Journal: Nucleic Acids Research

Article Title: Spatially multiplexed RNA in situ hybridization to reveal tumor heterogeneity

doi: 10.1093/nar/gkz1151

Figure Lengend Snippet: Differential HER2 gene expression in mammary carcinoma sections. ( A ) Integrated RNA-ISH fluorescence intensity per cell section for ActB and HER2 in a mammary carcinoma section (lower image). Cohen effect sizes describing interregional differences in expression levels calculated using a Mann–Whitney test are shown as matrices for ActB and HER2 , respectively (upper images). ( B ) Histological overview of the tumor tissue section stained with hematoxylin and eosin (upper image) and HER2 immunohistochemistry (lower image) with the regions 1–4 used for local RNA-ISH detection in (C). Scale bar: 1 mm. ( C ) Integrated RNA-ISH fluorescence intensity ( HER2 ) per cell section for four areas in each of the four different tumor regions in a mammary carcinoma section with a HER2 IHC status of 3+. The entire tissue sections including the regions of interest are shown as hematoxylin and IHC stained tissues in . The Cohen effect size ( r ) for interregional heterogeneity was calculated from all areas within each region combined by using a Mann-Whitney test and its values are displayed above the graph. ( D ) Quantitative immunohistochemistry using a gradient of primary antibody incubation times by local antibody delivery . The graph shows the relative mean intensity of the diaminobenzidine (DAB) signal after the amplification of the secondary antibody signal through a horseradish peroxidase reaction versus the incubation time of the primary antibody. It provides a quantitative measure of protein expression levels.

Article Snippet: Breast carcinoma tissue sections were either obtained from BioChain (T2235086) or were kindly provided and anonymized by the tissue biobank of the Department of Pathology and Molecular Pathology of the Universitätsspital Zürich.

Techniques: Expressing, Fluorescence, MANN-WHITNEY, Staining, Immunohistochemistry, Incubation, Amplification