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Results: 4

1.
Figure 1

Figure 1. From: Quantitative In Situ Detection of Phosphoproteins in Fixed Tissues Using Quantum Dot Technology.

Validation of quantum dot (QD) immunofluorescence (IF) quantitation of phosphoproteins (PPs). UT7/erythropoietin (EPO) or HT-29 cells were treated with EPO or H2O2/IFG-1, respectively, to modulate PP levels. Paired samples were subjected to lysis or 10% formalin-fixed paraffin-embedded cell block preparation. Western blot (WB) quantitation of phospho-STAT5 (pSTAT5; Y694/99) in the UT7/EPO cell model or of pAKT (S473) in the HT-29 cell model was correlated with QD IF quantitation of corresponding cell blocks. Axes are expressed as relative units of optical density vs relative combined score. The Pearson's correlation coefficients in both models were at least 0.93 (p<0.001). Data presented are representative of at least five independent experiments.

Juraj Bodo, et al. J Histochem Cytochem. 2009 July;57(7):701-708.
2.
Figure 3

Figure 3. From: Quantitative In Situ Detection of Phosphoproteins in Fixed Tissues Using Quantum Dot Technology.

Evaluation of the expression of different PPs using the Kinetworks PP screen in the panel of various human cell lines. PC 3 (prostate adenocarcinoma), MCF-7 (mammary gland adenocarcinoma), HT-29 and HCT 116 (colorectal adenocarcinoma), HH and Hut 78 (cutaneous T-cell lymphoma), HEL (erythroleukemia) and UT7/EPO (acute myelogenous leukemia), Karpas 299 (anaplastic T-cell lymphoma), SUDHL6 (B-cell lymphoma) and Raji (Burkitt's lymphoma), and K562 (chronic myeloid leukemia). PPs are normalized to the cell line with the highest expression of corresponding PP, and relative intensities are shown on the heat map on a 0–1 linear scale. The color scale is based on the optical spectrum. Red indicates increased PP level, yellow represents 50% intensity of the highest expressed cell line, green indicates decreased PP level, and black represents no detectable expression.

Juraj Bodo, et al. J Histochem Cytochem. 2009 July;57(7):701-708.
3.
Figure 4

Figure 4. From: Quantitative In Situ Detection of Phosphoproteins in Fixed Tissues Using Quantum Dot Technology.

Application of standardized QD quantitation in therapy-monitoring and diagnostic models. (A) Oral dosing with enzastaurin decreases GSK3β (S9) phosphorylation. Nude mice bearing HCT116 xenografts (1-cm diameter) were treated with 75 mg/kg enzastaurin for 10 days twice daily by gavage. On day 10 of dosing, mice were killed, and tumors were harvested at 0.5, 2, and 4 hr after the last dose. As a control, tumors from untreated mice were used. As the standard, MCF-7 was used as the cell line with the highest expression, HH as the cell line with medium expression, and UT7/EPO as the cell line with no expression of phospho-GSK3β. (B) ITD positivity in an acute myeloid leukemia (AML) patient increases nuclear STAT5 (Y694/99) phosphorylation. Patient samples were divided into two groups, ITD-positive cases (C1–C6) and ITD-negative cases (C7–C14). Statistical significance and t-test are shown. K562 cells served as the standard with the highest expression, HH as the cell line with medium expression, and MCF-7 as the cell line with no expression of pSTAT5 (Y694/99). The data are expressed as mean ± standard deviation.

Juraj Bodo, et al. J Histochem Cytochem. 2009 July;57(7):701-708.
4.
Figure 2

Figure 2. From: Quantitative In Situ Detection of Phosphoproteins in Fixed Tissues Using Quantum Dot Technology.

Optimization of fixation process. (A) Effects of delay in freezing on PP levels in xenograft tumors. Immediately after the harvest, tumors were frozen in liquid nitrogen or incubated in PBS at room temperature for the indicated times and subsequently dried and frozen. WB analyses of different PPs in HCT116 and pSTAT5 (Y694/99) in HEL mouse xenografts showed significant changes in PP levels when delay in freezing occurred. (B) Performance comparison of five commercially available fixatives: 10% buffered formalin, Zn formalin, B5, UMFIX (Tissue-Tek Xpress), and Prefer, in HEL mouse xenografts stained with anti-pSTAT5 (Y694/99) antibody. The data represents five independent experiments. A t-test was performed and statistical significance from the data of samples fixed in the 10% formalin is shown. *p<0.05, **p<0.01. (C) WB analysis of pAKT (S473) in HT-29 mouse xenografts shows decrease in signal after LR3-IGF-1 treatment. QD-based IF of paraffin-embedded HT-29 mouse xenografts illustrates varying intensities of cytoplasmic pAKT (S473) staining. Numbers correspond to lanes of the WB. WB and QD IF quantitation of paired tumor sections shows good correlation between pAKT (S473) levels in LR3-IGF-1-stimulated HT29 xenografts. Tumors were harvested and processed immediately or held in PBS for 2 hr. The data presented are representative of at least four independent experiments. The data are expressed as mean ± standard deviation.

Juraj Bodo, et al. J Histochem Cytochem. 2009 July;57(7):701-708.

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