To detect Akh expression in the neuroendocrine cells of adult flies, Akh RNAi transgenic flies
or w-control flies were crossed against Akh-Gal4 driver flies and, after hatching, the virgin
female progeny was kept for five days at 25°C. At this time, the corpora cardiac (cc) and
attached tissues were manually dissected in 1×PBS. After fixation with 4% paraformaldehyde
for 2 h on ice, tissues were washed 20 min in 1×PBS and 2×20 min in TNT (0.1 mol/L Tris
HCL pH 7.5, 0.3 mol/L NaCl, 0.5% TritonX-100), and then blocked in TNT + 4% NGS
(normal goat serum) for 1 h at room temperature. Tissues were incubated at 4°C overnight
with -Akh antibody (1:300, Kim and Rulifson, 2004) and mouse -GFP antibody (1:600, A11121, Invitrogen/Thermo Fisher Scientific, USA), and washed in 1×PBST (PBS + 0.6%
TritonX-100) for 3×10 min at room temperature before incubation with secondary antibodies
(Alexa-568-goat anti-rabbit, 1:1000, Invitrogen/Thermo Fisher Scientific; Alexa-488-goat mouse, 1:1000, Jackson ImmunoResearch Laboratories, USA) in TNT + 4% NGS for 2h at
room temperature. Samples were washed in 1×PBST for 3×10 minutes at room temperature
and kept overnight in 1×PBS at 4°C. For imaging, tissues were mounted in 60%
glycerol/1×PBS and analysed on a Zeiss LSM-780 microscope (Zeiss, Germany) at 25×
magnification in fixed gain mode. Maximum intensity projections of image stacks were done
using the ZEN 2010 software (Zeiss).
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Supplementary figure legend
Fig. S1. Stim, Plc21C, Gγ1, Gαq, AkhR and Akh gene regulation in response to transgenic
gene knockdown or gene overexpression at the transcriptional (A) and protein (B) level.
A: Transcriptional regulation of Plc21C, G1, Gq, AkhR, Akh and Stim genes in response to
fat body targeted (FB-Gal4) or ubiquitous (da-GS) RNAi-mediated in vivo knockdown or
overexpression (gof) detected by qRT-PCR analysis. B: Depletion of Akh peptide (red) from
the corpora cardiaca (cc, highlighted by GFP expression in green) in response to cc-targeted
Akh gene knockdown. Shown are maximum intensity projections of confocal fluorescence
images of -GFP (left panel) and -Akh (middle panel) immunohistochemistry. Arrows point
to the cell bodies of cc cells. Scale bars represent 20 µm.