Journal: Histopathology

Article Title: Cell-based quantification of molecular biomarkers in histopathology specimens

doi: 10.1111/j.1365-2559.2011.03878.x

Figure Lengend Snippet: Duplex analysis of phospho-extracellular signal-related kinase (p-ERK) and Ki67 immunostaining in human breast carcinoma cells. Sections of two different breast tumours were stained and analysed as described for Figure 5. Brightfield images of the two different tumours are shown (A,G), with the unmixed channels for 3,3-diaminobenzidine (Ki67) (B,H) and SG Blue (p-ERK) (C,I). Composite images showing whole-cell segmentation of the tumour (cytokeratin-positive) cells are shown (D,J). Scatter plots of p-ERK (x-axis) and Ki67 (y-axis) staining intensity are shown for tumour cells (E,K) and for non-tumour (stromal) cells (F,L), with each dot representing one cell.

Article Snippet: ER, PR, Ki67, p-ERK and HER2 were detected by immunohistochemistry with biotinylated species-specific secondary antibodies, avidin-linked horseradish peroxidase (HRP) (ABC Kit) and 3,3-diaminobenzidine or SG Blue (Vector Laboratories) HRP chromogen substrate.

Techniques: Immunostaining, Staining

Journal: Histopathology

Article Title: Cell-based quantification of molecular biomarkers in histopathology specimens

doi: 10.1111/j.1365-2559.2011.03878.x

Figure Lengend Snippet: Duplex analysis of phospho-extracellular signal-related kinase (p-ERK) and Ki67 immunostaining in lymphoid cells in a human breast carcinoma. A section of a breast tumour was stained sequentially with anti-p-ERK (SG Blue), anti-Ki67 [3,3-diaminobenzidine (DAB)] and anti-CK (Alexa-488) antibodies, and this was followed by haematoxylin staining, multispectral imaging (×400), and cytometric analysis. The brightfield image of a lymphoid nodule in the tumour is shown (A), along with the unmixed channels for DAB (Ki67) (B), SG Blue (p-ERK) (C), and Alexa-488 (cytokeratin) (D). Scatter plots of p-ERK (x-axis) and Ki67 (y-axis) staining intensity are shown for cells in the lymphoid nodule (E) and for tumour cells (F), with each dot representing one cell.

Article Snippet: ER, PR, Ki67, p-ERK and HER2 were detected by immunohistochemistry with biotinylated species-specific secondary antibodies, avidin-linked horseradish peroxidase (HRP) (ABC Kit) and 3,3-diaminobenzidine or SG Blue (Vector Laboratories) HRP chromogen substrate.

Techniques: Immunostaining, Staining, Imaging

Journal:

Article Title: In Situ Visualization of Intratumor Growth Factor Signaling

doi:

Figure Lengend Snippet: Immunohistochemical demonstration of ERK/MAPK phosphorylation in astrocytic neoplasms. For each tumor, adjacent sections were immunolabeled with the phospho-specific ERK/MAPK antibody (pMAPK; left panels ) and a phospho-insensitive ERK/MAPK antibody (MAPK; right panels). Elevated nuclear and cytoplasmic dp-ERK/MAPK immunoreactivity, relative to normal quiescent astrocytes, was detected in a subset of tumor cells within a variety of astrocytic neoplasms. In contrast, ERK/MAPK immunoreactivity was more uniformly expressed. A and B: Astrocytoma, WHO grade II. Some diffusely infiltrating astrocytoma cells show increased nuclear and cytoplasmic dp-ERK/MAPK immunoreactivity. Note the absence of labeling in adjacent normal neurons. ERK/MAPK protein is more evenly expressed in tumor cell cytoplasm and in neurons. C and D: Anaplastic astrocytoma, WHO grade III. A subset of gemistocytic tumor cells shows high cytoplasmic and nuclear dp-ERK/MAPK immunoreactivity. E and F: Ependymoma. A subpopulation of highly fibrillar ependymoma cells, especially those forming perivascular pseudorosettes, shows elevated dp-ERK/MAPK immunoreactivity; compare with the relatively uniform expression of ERK/MAPK protein. G and H: Glioblastoma multiforme (astrocytoma, WHO grade IV). Tumor cells near the hyperplastic microvasculature show the most intense nuclear and cytoplasmic pMAPK immunoreactivity. Some fine immunoreactive tumor cell processes appear to be oriented toward the microvasculature (corner inset). Total MAPK immunoreactivity is much more uniform, and not restricted to the perivascular tumor cells. I and J: Glioblastoma with giant cell transformation (astrocytoma, WHO grade IV). Only the large, bizarre, multinucleated tumor cells have detectable dp-ERK/MAPK immunoreactivity; the small cell population is completely negative. Note the absence of dp-ERK/MAPK immunoreactivity in a mitotic tumor cell (left side). In contrast, both small and large tumor cell populations show uniform ERK/MAPK protein immunoreactivity. Also, a mitotic cell is visible that has detectable MAPK immunoreactivity (right side). K to N: Selective dephosphorylation of ERK/MAPK within mitotic and cycling glioblastoma cells. K and L: Immunoreactivity for dp-ERK/MAPK is absent within individual mitotic tumor cells in two additional glioblastoma specimens; adjacent interphase cells show abundant cytoplasmic and nuclear immunoreactivity. M: Evidence that mitotic phosphoepitopes in general are not lost in routinely processed surgical specimens comes from immunolabeling with monoclonal antibody MPM-2, which recognizes a number of mitosis-specific phosphorylated proteins. Strong perichromosomal staining is present in mitotic cells. N: Double-label immunohistochemistry for dp-ERK/MAPK (red-brown) and Ki-67 (dark blue) reveals essentially complete non-co-localization. This suggests that cycling cells (those with nuclear Ki-67 immunolabeling) and those with activated ERK/MAPK (dp-ERK/MAPK immunoreactive) are generally mutually exclusive populations. A to M: Immunoperoxidase staining (DAB) with hematoxylin counterstain. N: Double-label immunoperoxidase with aminoethylcarbazole and Vector SG substrates; no counterstain. Scale bar in A represents 50 μm and applies to A to J and M; bar in N represents 20 μm and applies to K, L, and N.

Article Snippet: Double-label immunohistochemistry was performed sequentially using the peroxidase substrates aminoethylcarbazole and Vector SG, according to the manufacturer’s instructions (Vector Laboratories, Burlingame, CA).

Techniques: Immunohistochemical staining, Immunolabeling, Labeling, Expressing, Transformation Assay, De-Phosphorylation Assay, Staining, Immunohistochemistry, Immunoperoxidase Staining, Plasmid Preparation