Anti prospero

The previously published gut immunocytochemistry protocol was used [16 (link)] with minor modifications. Briefly, whole abdomens were immunostained before the intestines were dissected out and mounted for imaging. Whole abdomens were separated and punctured for reagent infiltration to the intestine in phosphate buffered saline with 0.2% Triton X-100 (PBS-T, pH 7.2), and fixed in 4% paraformaldehyde in PBS-T overnight at 4°C. After 3 washes with PBS-T, samples were blocked with 3% goat serum in PBS-T for at least 30 min. Abdomens were incubated with primary antibodies or antisera for 1–2 days at 4°C, washed with PBS-T, and incubated with secondary antibodies overnight at 4°C. The primary antibodies used were rat anti-TRPA1 (1:200) [19 (link),22 (link),37 (link)], rabbit anti-GFP (1:1000, Life Technologies, CA, USA), anti-Prospero (1:10, Developmental Studies Hybridoma Bank at the University of Iowa). The secondary antibodies used were Alexa Fluor Cy3-labeled goat anti-rat (1:1000, Jackson Laboratory, ME, USA), Alexa Fluor 488-labeled goat anti-rabbit (1:200, Life Technologies, CA, USA), and Alexa Fluor 568-labeled goat anti-mouse IgG (1:1000, Life Technologies, CA, USA). A Zeiss LSM 700 laser-scanning confocal microscope was used to acquire images of immunostained samples.

For immunofluorescence staining, intestines were dissected in phosphate-buffered saline (PBS) and fixed in 8% paraformaldehyde for 3 hours. Tissues were washed with 0.1% Triton X-100 in PBS and blocked in 1% bovine serum albumin for 1 hour. Subsequently, tissues were stained with anti–β-galactosidase (1:400) (MP Biomedicals, catalog no. 0855976-CF), anti-Prospero (1:1000) (Developmental Studies Hybridoma Bank, MR1A), anti-p4EBP (Cell Signaling Technology, 2855) antibodies. The samples were mounted in Vectashield mounting medium with DAPI (Vector Laboratories).

Primary antibodies used included anti-deadpan (Abcam ab195174; 1:100), anti-GFP (Invitrogen A10262; 1:200), anti-DCP1 (Cell Signaling 9578; 1:200), anti-phospho-histone H3 ser10 (Cell Signaling 9701; 1:500), anti-prospero (Developmental Studies Hybridoma Bank MR1A; 1:1000, and anti-mCherry (Cell signaling 43590; 1:200). All secondary antibodies were used at a dilution of 1:500 and included Goat anti-Chicken IgY (H + L) Secondary Antibody, Alexa Fluor™ 488 (Thermo Fisher A-11039), Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 568 (Thermo Fisher A-11011), Donkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 647 (Thermo Fisher A-31573), Donkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488 (Thermo Fisher A-21206), Goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488 (Thermo Fisher A-11001), Goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 568 (Thermo Fisher A-11004), and Goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 647 (Thermo Fisher A-21235).