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. 2011 Jan;59(1):76–87. doi: 10.1369/jhc.2010.955948

Figure 4.

Figure 4.

(A) Comparison of fluorescence intensity of nuclear pore complex protein (NPC) staining in human fibroblast cells using fluorescent polymerization-based amplification (FPBA; light gray), tyramide signal amplification (TSA; dark gray), or SA–Alexa 488 (black). The p-values for a one-tailed Student’s t-test between FPBA and TSA were p1 = 1.1 × 10−3, p2 = 3.4 × 10−2, p3 = 2.7 × 10−11, p4 = 1.1 × 10−3, and p5 = 1.9 × 10−1. Although p-values were determined for all data sets, it is of note that the data sets for the no-primary antibody condition were not normally distributed, nor were the FPBA data for the 1:100 antibody dilution. *Indicates that most of these signal intensities were saturated. **Indicates that these measurements were not taken. (B) Nonspecific staining (“noise”) was assessed in each cell by measuring the fluorescence in an area equal to the size of the nucleus but immediately adjacent to the nucleus. For each cell, the intensity of staining in the nucleus (“signal”) was divided by the noise value. The ratio of signal/noise was compared between FPBA (light gray) and TSA (dark gray). Although these data sets were not normally distributed, p-values for a one-tailed t-test were determined: p1 = 8.5 × 10−2, p2 = 6.0 × 10−2, p3 = 1.0 × 10−4, p4 = 3.6 × 10−3. For both A and B, measurements were taken for human fibroblast cells stained under the same staining and imaging conditions as used for the images in Figure 3. Also, for each primary antibody dilution in A and B, measurements were made for all cells in each of four images obtained from at least two separate staining sessions; however, when it was not possible to take a noise measurement immediately adjacent to the nucleus, that cell was not included in the signal/noise measurements. For A and B, intercellular background signal measured far from the cells was subtracted from the measured signal and noise intensities. DAPI (4′,6-diamidino-2-phenylindole dihydrochloride) staining was used to delineate areas that were expected to be stained positive for NPC.