FIGURE 2.

Linearity of antibody staining. (A–D) A single embryo was fixed in formaldehyde during nuclear cycle 14 (6.7% paraformaldehyde in 1× phosphate-buffered saline for 45 min) and stained with rabbit anti-GFP primary antibody (Millipore [previously Chemicon], Billerica, MA) following published protocols (Wieschaus and Nüsslein-Volhard 1986). The secondary antibody was conjugated with infrared Alexa-647 (Molecular Probes/Invitrogen Corporation, Carlsbad, CA), maximally reducing spectral overlap with the green GFP autofluorescence. The embryo was imaged both at the surface (A, C) and at the midsagittal plane (B, D) using confocal microscopy (Leica SP5, 20× oilimmersion objective plan apochromat, NA = 0.7; Leica Microsystems, Germany). GFP-autofluorescence (A, B) and anti-GFP staining (C, D) were recorded in consecutive runs. Scale bars, 100 µm. Typical dimensions of Drosophila eggs are 500 × 1800 µm. (E) Extracted raw fluorescence intensity profiles from (A) and (C) projected on the embryo’s anteroposterior axis. Each point corresponds to a single nucleus (for details, see Gregor et al. 2007b). (F) Raw fluorescence intensity profiles from (B) and (D) projected on the embryo’s anteroposterior axis, extracted by sliding a circular averaging area along the edge of the embryo (for details, see Houchmandzadeh et al. 2002). (G) Scatter plot of GFP-autofluorescence intensities versus fluorescence antibody staining intensities extracted from (A) and (C) for all nuclei. Each point corresponds to a single nucleus; the curves are normalized by nuclei of maximal and minimal intensities. (H) Scatter plot of fluorescence intensities extracted from (B) and (D). The blue line corresponds to the dorsal profile and the red line corresponds to the ventral profile.