Fig 1: Reaction kinetics can be quantified in well-defined synthetic antigen controls. Shown are representative images of anti-BCL2-AlexaFluor™-647 antibody binding to tissue microarray cores with 2.5E-5 M BCL2 peptide (A-D) and control cores lacking peptide (E-H) after antigen retrieval at 95°C and incubation with three successive [Ab]: (A,E) 9.1E-10 M @ 48 hr; (B, F) 2.5E-9 M @ 68 hr; (C,D,G,H) 9.8E-9 M @ 85 hr. Images A-C, E-G are Cy5 channel, 1 s exposures. Images D and H are DAPI channel (autofluorescence), 0.04 s exposure. All images are shown equally scaled at 0-32768 fluorescent units. Circular regions of interest (ROIs 01–12, top left to lower right) are 100 pixel (38μm) in diameter. Cis-glass control ROIs (n = 7 for Ag-containing cores; n = 4 for control cores) are 70 pixel (26.4μm) squares. (I,J) Observed IHC reaction time courses for ROI 01 (circles) and ROI 12 (squares) are plotted on linear and log scales; data are means ± 2 SD of 420–588 unique background-corrected [AgAb] values at each timepoint; on average, 3.8% of ROI values at each timepoint were programmatically excluded as outliers relative to other values. Horizontal lines (I) are [AgAb] plateau values within 2 SD of the mean consistent with: solid black, nominal observed data; red, minimum time course fitting error; blue, minimum Langmuir equilibrium fitting error; black dashed, KD max / [Ag] max; dash-dot, KD min; dotted, [Ag] min. Curves (J) are fitted time courses; contour boundaries (K) are 3x the minimum fitting error of the optimum parameter pairs (circles, ROI 01; squares, ROI 12). Heat maps (L, N) show the average fitting error for the overlapping contour plots (K) for ROIs 1 and 12, respectively, and for ROIs 2–11 (M). Color map values are −log10(average fitting error). Fitting data for ROIs 1–12 are summarized in Table S6.
Fig 2: Time to IHC reaction equilibrium correlates with [Ag] in diffusion-limited reactions. Anti-BCL2 Ab binding kinetics measured in synthetic antigen control samples containing BCL2 peptide at 2.5E-6 M (circles; A,C,D,F), 2.5E-5 M(squares; B,C,E,F) or 2.5E-4M (diamonds; C, F). FFPE sections were stained with AlexFluor™-647-conjugated anti-BCL2 clone 124 at successively increasing [Ab]: 1 nM (0-1.8E5 s), 3 nM (1.8-2.4E5 s), and 10 nM (2.4-3.1E5 s) following antigen retrieval at 99°C. (C, F) Time course for samples with all three [Ag] are plotted for comparison. Plotted data are the means ± 2 SD of unique values (n=54–144) from combinatorial nonspecific signal corrections. Points on the x-axis (A, B, C) are programmatically excluded as outliers relative to adjacent timepoints, or due to saturated pixels (see Methods).
Supplier Page from Novus Biologicals, a Bio-Techne Brand for Bcl-2 Antibody (124) [Alexa Fluor® 647]
Available conjugates: Available conjugates: Alexa Fluor 647Specificity: This antibody recognizes a protein of 25-26kDa, identified as the Bcl-2 alpha oncoprotein. It shows no cross-reaction with Bcl-x or Bax protein. Expression of Bcl-2 alpha oncoprotein inhibits the programmed cell death (apoptosis). In most follicular lymphomas, neoplastic germinal centers express high levels of Bcl-2 alpha protein, whereas the normal or hyperplastic germinal centers are negative. Consequently, this antibody is valuable when distinguishing between reactive and neoplastic follicular proliferation in lymph node biopsies. It may also be used in distinguishing between those follicular lymphomas that express Bcl-2 protein and the small number in which the neoplastic cells are Bcl-2 negative.Sizes Available: 0.1 ml