Prolong gold antifade mounting with dapi

For immunofluorescence, organoids were seeded as single cells on a Falcon 4-well Culture slide (FAL354114, In Vitro Technologies, Mt Wellington, Auckland, New Zealand) with 200 cells per well and were induced at day 1 with endoxifen as previously described. If required, organoids were drugged at day 2 for 48 h. At day 4 or 5, organoids were washed for 1 h with PBS and fixed in 1% PFA for 1 h, followed by blocking in 10% FHS/0.5% Triton in PBS. Organoids were then incubated for 3 h, at RT, with primary antibody E-Cadherin 1:100 (AF748, R&D Systems, Minneapolis, MN, USA), Tp53 1:100 (ab246550, Abcam, Cambridge, UK) or Ki-67 1:100 (ab1667, Abcam, Cambridge, UK), followed by a 2 h incubation with the according secondary antibody Donkey anti-Goat Alexa fluor 488 1:400 (A11055, Thermo Fisher Scientific, Waltham, MA, USA) or Goat anti-Rabbit Alexa Fluor 488 1:500 (A11008, Thermo Fisher Scientific, Waltham, MA, USA). Slides were then mounted with ProLong Gold Antifade Mounting with DAPI (P36935, Thermo Fisher Scientific, Waltham, MA, USA) and imaged with a Nikon A1+ inverted confocal laser scanning microscope and a Nikon DSiR2 color 16 MP camera (Otago Micro and Nanoscale Imaging, University of Otago). Organoids were imaged every 10 microns.

FFPE skin blocks were cut into 6 μm sections and placed on slides. Sections were first deparaffinized and rehydrated, then Heat-Induced Epitope Retrieval (HIER) was performed and sections were permeabilized with PBS 0.01% Triton. Samples were stained for 2 h at room temperature with the following primary antibodies: goat anti-human IL-26/AK155 (R&D systems, #AF1375, 1/100), mouse anti-human TGF beta-1 (Thermo Fisher Scientific, # MA5-16949, 1/500), rabbit anti-human CD3 (Ventana, # 790-4341, ready to use). Sections were then stained for 30 min at room temperature with the following fluorescently-labeled secondary antibodies: donkey anti-rabbit IgG (H + L) Alexa Fluor 488 (Invitrogen, #A-32790, 1/500), chicken anti-mouse IgG (H + L) Alexa Fluor 647 (Invitrogen, #A-21463, 1/500), donkey anti-goat IgG (H + L) Alexa Fluor 546 (Invitrogen, #A-32790, 1/500). For RNA ISH, TGFB1 mRNA (#400881) was detected in skin using RNAScope® Multiplex Fluorescent V2 Assay following the manufacturer’s instruction (Advanced Cell Diagnostics, Inc). Sections were then labeled with OPAL 650 (Akoya Biosciences, #OP-001005, 1/1500). All slides were mounted with ProLong Gold antifade mounting with DAPI (Thermo Fisher Scientific). Images were acquired with a Zeiss LSM 700 confocal microscope and analyzed with Zen 2010 software.

Chromosomal preparations were incubated in the 2 × SSC with 4% formaldehyde solution at 60 °C for 30 min. Then, 15 μl of the labeled GAL4-specific fluorescent DNA-probe dissolved in the hybridization buffer were applied to the slide and covered with a 22 × 22 coverslip. The borders of the coverslip were insulated with rubber cement. Slides were then placed into the Thermobrite machine (Leica Biosystems, Wetzlar, Germany) and heated at 80 °C for 5 min to denature chromosomal DNA and the probe. Hybridization occurred at 37 °C overnight. On the next day, the slides were washed in 2 × SSC at 60 °C for 15 min, then in 2 × SSC at room temperature for 15 min, and finally in 0.2 × SSC for 10 min. The washing solution was removed from the slide, and 15 μl of Prolong Gold Antifade Mounting with DAPI (ThermoFisher Scientific, Waltham, MA, USA) was applied to the slide and covered with a 22 × 22 coverslip. Microscopy and image acquisition were performed with an Axio Imager Z1 microscope (Carl Zeiss MicroImaging GmbH, Munich, Germany). Image processing was performed by the Fiji software (cite: doi:10.1038/nmeth.2019). Mapping of the location of the probe was done according to the standard cytogenetic map of polytene chromosomes for An. stephensi^{24 (link)}.