Triton χ 100

Transthyretin (TTR) is the first known protein
synthesized solely by the CP and is a marker for
CPECs. In this study, for confirmation that cells isolated
from the brain are CPECs and not another cell, the
immunocytofluorescence was performed to identify the
TTR marker. Immunocytofluorescence technique was
performed according to the method previously described
(22 (link)). A 24-well plate was used to CPECs culture. Then,
cells were fixed with 4% paraformaldehyde (Sigma,
USA). The next steps were the washing with PBS, to
be permeable with Triton Χ-100 (Sigma, USA), and the
incubation with the normal goat serum (Abcam, England).
Afterward, cells were incubated with the primary
antibody against TTR at 4˚C. After washing with PBS,
the fluorescent secondary antibody (Abcam, England)
was used. Finally, the cells were painted by diamidino-2-
phenylindole (DAPI) dye (Sigma, USA) and studied by
fluorescence microscopy (Olympus, IX 71, Japan).

This method was performed to exclude differences in food intake between control flies and radish sprouts treated flies. The gustatory assay was performed as described earlier [34 (link)]. In brief, 15 flies were kept on SY medium or SY+radish sprouts. Both were supplemented with 0.2% w/v sulforhodamine B sodium salt (Sigma-Aldrich, Steinheim, Germany) and kept under standard conditions for 500 min. Next, the flies were homogenized in PBS (Life Technologies by Thermo Fisher Scientific, Darmstadt, Germany) plus 1% Triton™ Χ-100 (Sigma-Aldrich) using a Qiagen TissueLyser II (Hilden, Germany). The flies were then centrifuged, and the absorbance was measured in an Infinite 200 spectrophotometer (Tecan, Crailsheim, Germany) at 535/25 nm excitation and 590/20 nm emission wavelength.