The following antibodies were used for immunohistochemistry in this study: mouse anti-Scr (1:50; 6H-4.1, DSHB), mouse anti-Ubx (1:40; FP3-38, DSHB), mouse anti-Abd-A (1:100; 6A8.12, DSHB), mouse anti-Abd-B (1:40; 1A2E9, DSHB), mouse anti-EcR-B1 (1:50; AD4.4, DSHB), mouse anti-FasII (1:50; 1D4, DSHB), mouse anti-Cut (1:50; 2B10, DSHB), mouse anti-Knot/Collier (1:100, a gift from A. Vincent), Guinea pig anti-Sox14 (1:200), Guinea pig anti-Mical (1:500), Rabbit anti-GFP (1:1000, A-11122, Invitrogen), Rabbit anti-Scm (1:20; a gift from J. Muller). Fluorescein isothiocyanate (FITC)-, Cy3- and Cy5-conjugated secondary antibodies (111–545-003, 115–165-003, 111–165-003, 106–165-003 and 123–605-021, Jackson ImmunoResearch) were used at 1:500 dilution.
For immunohistochemistry, pupae or larvae were dissected in cold PBS and fixed in 4% formaldehyde for 20 min, followed by washing with 0.5% Triton-X-containing PBS (PBST) for 3 times. Control and sample fillets/brains for each set of experiment were washed and stained in the same tube. Primary antibodies were added into blocking buffer (5% BSA in PBST) after 30 min blocking and were incubated at 4 °C overnight. Secondary antibodies were incubated on the second day at room temperature for 2–6 h. Samples were mounted using VectaShield mounting medium and imaged using either Leica SPE II or Olympus FV3000 confocal microscope. Images were taken from projected z-stacks (1.5-µm intervals) to cover the whole da neuron. Images of the same experiment set were taken with the same settings and processed in parallel.
Measurement of fluorescence intensity was done using ImageJ. Contours of cell nuclei (Ubx/ Abd-A/ Abd-B/ Scr/ EcR-B1/ Sox14/ Cut/ Knot immunostaining) or whole soma (Mical immunostaining) were drawn on the GFP channel. To quantify the fluorescence intensity of Scr, Cut and Knot background (rolling ball radius = 50) was subtracted on the whole image of that channel before measuring the mean grey value in the marked region of ddaC nuclei was measured. To quantify the fluorescence intensity of Ubx, Abd-A, EcR-B1, Sox14 and Mical, background (rolling ball radius = 50) was subtracted on the whole image of that channel before measuring the mean grey value in the marked region of ddaC and ddaE, their ratio was subsequently calculated. The values were then normalized to their corresponding mean control values and subjected to statistical analysis.
For immunohistochemistry, pupae or larvae were dissected in cold PBS and fixed in 4% formaldehyde for 20 min, followed by washing with 0.5% Triton-X-containing PBS (PBST) for 3 times. Control and sample fillets/brains for each set of experiment were washed and stained in the same tube. Primary antibodies were added into blocking buffer (5% BSA in PBST) after 30 min blocking and were incubated at 4 °C overnight. Secondary antibodies were incubated on the second day at room temperature for 2–6 h. Samples were mounted using VectaShield mounting medium and imaged using either Leica SPE II or Olympus FV3000 confocal microscope. Images were taken from projected z-stacks (1.5-µm intervals) to cover the whole da neuron. Images of the same experiment set were taken with the same settings and processed in parallel.
Measurement of fluorescence intensity was done using ImageJ. Contours of cell nuclei (Ubx/ Abd-A/ Abd-B/ Scr/ EcR-B1/ Sox14/ Cut/ Knot immunostaining) or whole soma (Mical immunostaining) were drawn on the GFP channel. To quantify the fluorescence intensity of Scr, Cut and Knot background (rolling ball radius = 50) was subtracted on the whole image of that channel before measuring the mean grey value in the marked region of ddaC nuclei was measured. To quantify the fluorescence intensity of Ubx, Abd-A, EcR-B1, Sox14 and Mical, background (rolling ball radius = 50) was subtracted on the whole image of that channel before measuring the mean grey value in the marked region of ddaC and ddaE, their ratio was subsequently calculated. The values were then normalized to their corresponding mean control values and subjected to statistical analysis.
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