Anti gfp

Heads and thoraxes from adult flies were dissected and fixed in 4% paraformaldehyde in phosphate buffer saline (pH7.2) overnight at 4°C, followed by cryoprotection with 30% sucrose for 48 h at 4°C (heads were first pre-incubated with 10% sucrose for 2 h). Then they were embedded in OCT compound and cut in the cryostat (16 μm for thoraxes and 14 μm for heads). Cryosections were washed in PBT, blocked (PBS containing 5% BSA and 0.3% Triton X-100) for 45 minutes at room temperature and incubated with anti-GFP (Rockland) overnight at 4°C. After washes with PBT, the tissue was incubated with secondary antibodies. After washes with PBT, cryosections, the tissues were incubated with phalloidin-rhodamine diluted in PBT and then with DAPI. Sections were mounted with Aqua Poly/mount (Polysciences, Inc., Warrington, PA). Images were obtained with a Leica SP8 microscope.

Anti-BrdU (1∶200, Millipore, MAB3424), anti-E-Cadherin (1:500, BD Biosciences, 610181), anti-GFP (1:1000, Rockland, 600-101-215), anti-phospho myosin light chain 2 (1:300, Cell Signaling Technologies, 3674), anti-Prickle1 antibody (1:300, Proteintech, 22589–1-AP), and anti-GFP-HRP antibody (1:200, Abcam, 600-101-215), anti-ZO1 antibody (1:300, Thermo Fisher Scientific, 33–9100).

ELISA plates were coated with the recombinant protein (5 μg/mL) in coating buffer (carbonate-bicarbonate buffer, pH 9.6; Medicago) overnight at 4°C and blocked with 5% skim milk in PBST for 30 min at 37°C. After rinsing with PBS, the analyte of recombinant protein was diluted in PBST (10, 100, or 1,000 nM) for 1 h at 37°C followed by three washes with PBST. Then, plates were treated with the primary antibodies (anti-TSG101 [catalog no. 28283-1-AP; Proteintech], anti-Alix [catalog no. 92880; Cell Signaling], and anti-GFP [catalog no. 600-101-215; Rockland]) for 1h at 37°C and washed 3 times with PBST, followed by the 1-h, 37°C incubation with the HRP-conjugated secondary antibody (HRP-anti-rabbit IgG [catalog no. 7074; Cell Signaling] and HRP-anti-goat IgG [catalog no. sc-2020; Santa Cruz]) and washed 3 times with PBST. The 1-Step Ultra TMB-ELISA substrate (Thermo) was added, and the reaction was stopped by 2 M H₂SO₄. The optical density at 450 nm (OD₄₅₀) was measured by an iMark microplate reader (Bio-Rad).