Fluorescently labeled secondary antibody

For immunofluorescence, the cells were sequentially probed with primary antibodies and fluorescently-labeled secondary antibodies (Jackson Immunoresearch, West Grove, PA, USA). Images were captured using a confocal microscope (FV3000; Olympus, Tokyo, Japan).

Mouse anti-GFP (mix of clones 7.1 and 13.1, Roche) (1:1000), rabbit anti-Vps35 (GTX108058, GeneTex) (1:1000), mouse anti-EGFR antibody (BD Transduction Laboratories) (1:200), HRP-labeled secondary antibodies (Sigma-Aldrich) (1:5000), rabbit-anti EEA1 antibody (Enzo) (1:100), fluorescently labeled secondary antibodies (Jackson ImmunoResearch Laboratories) (1:200), Hoechst (1:2500), Human EGF (Sigma). Mouse antibodies against SNX3 (C-16 Santa Cruz Biotechnology) (1:200).

Wandering third-instar larval eye discs were dissected in 1× PBS and fixed in 4% formaldehyde+1× PBS for 15 min, permeabilized in 0.3% PBS-T (1× PBS, 0.3% Triton X-100) two times for 10 min and then incubated with antibodies overnight at 4 °C in 1× PBS+10% normal donkey serum (NDS, Jackson Immunoresearch)+0.1% Triton X-100 blocking serum. The following day, samples were washed in 0.1% PBS-T (1× PBS+0.1% Triton X-100) three times for 5 min. Samples were then incubated with appropriate fluorescently labeled secondary antibodies (Jackson Immunoresearch) for 1 h in 1× PBS+10% NDS+0.1% Triton X-100 followed by 4′,6-diamidino-2-phenylindole (DAPI) for 5 min. Finally, samples were washed five times for 5 min then mounted in FluorSave (EMD Millipore) on glass slides. All steps were carried out at RT and with gentle rocking, unless specified otherwise. Whenever fluorescent images have been compared, they have been obtained with the same acquisition and display settings.
Primary antibodies used were: anti-β-gal: DSHB 40-1a (1:200), anti-cleaved Drosophila Dcp-1: Cell Signaling Asp216 (1:500), anti-Dac DSHB mAbdac1-1 (1:100), anti-Elav: DSHB 7E8A10 (1:200), anti-GFP (FITC): Abcam ab6662 (1/1000), anti-Hairy: from T. Orenic (1:4), anti-Hth: from Richard Mann (1:2000), anti-Notch DSHB C458.2H (1:100), anti-Repo: DSHB 8D13 (1:500), anti-Senseless: from H. Bellen (1:100).

Sections from cryo-preserved human foreskin or 3D human skin equivalents and freshly isolated primary human keratinocytes were used. Antibodies detecting nuclear (active) phospho-NRF2 (S40) (Abcam, EP1809Y) or p63 (Abcam, clone 4A4) were applied overnight following 4% PFA fixation of the specimens. K10 and K14 antibodies were from DAKO, and the Ki-67 antibody (SolA15) was from eBioscience (Thermo Fisher Scientific). Fluorescently labeled secondary antibodies (Jackson ImmunoResearch) were used. Nuclei were counterstained with DAPI. Cells were counted using ImageJ (National Institutes of Health). p63- and pNRF2-stained sections were analyzed by confocal microscopy. Deconvolution of confocal images was performed using Leica software.

Sections of kidney tissue were deparaffinized and antigen retrieval performed in citrate buffer. Tissue was incubated overnight at 4 °C in primary antibody directed against CD3 (1:100, R&D Systems, Minneapolis, MN, USA) or F4/80 (1:100, Bio-Rad Laboratories, Hercules, CA, USA). Fluorescently labeled secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) were applied the following day at room temperature for one hour, nuclei were stained with DAPI, and coverslips were mounted using ProLong Gold Antifade Mountant (Thermo Fisher, Waltham, MA, USA). Image stacks were taken on a Fluoview-1000 microscope with two to three regions examined per kidney. ImageJ [38 ] was used to threshold the channel of interest and the positive stained area was calculated and averaged across the images.

Frozen brain sections of 10 μm thickness were incubated with blocking buffer [10% normal goat serum (Thermo Fisher, Waltham, MA, United States) + 0.2% Triton X-100] for 1 h at RT. The sections were then incubated with primary antibodies (listed in Supplementary Table 1) at 4°C overnight. The sections were washed four times with 1× PBS at RT, each wash lasting 15 min. Subsequently, the brain sections were incubated with fluorescently labeled secondary antibodies (Jackson ImmunoResearch, West Grove, PA, United States) for 2 h at RT. Brain sections were again washed four times with 1× PBS at RT, as described before. To stain the nuclei, the brain tissues were mounted with antifade medium containing DAPI (Solarbio, Beijing, China), and observed under a fluorescence microscope (Olympus, CKX53SF, Japan). ImageJ software was used to quantify the immunofluorescence signal area, which was normalized to the area of CD31 signal.