Dibutylphthalate polystyrene xylene (dpx)

We performed single-color immunohistochemistry (IHC) for β-catenin in 5 μm-thick sections obtained from formalin-fixed, paraffin-embedded melanoma craniotomy tissues placed on positively charged glass slides, as we have previously described [26 (link)]. Briefly, slides were dried, then baked at 60 °C for 90 min, followed by heat-induced epitope retrieval using HIER Buffer L (Thermo Scientific, TA-135-HBL, Thermo Fisher Scientific, MA). Endogenous peroxidases were blocked using 3% hydrogen peroxide for 10 min at room temperature (RT). Tissues were then blocked using 10% normal goat serum for 1 h at RT and incubated with an antibody against β-catenin (rabbit monoclonal, clone 247, ab32572, 1:500 dilution, Abcam, MA) overnight at 4 °C. For negative control, we stained representative tissue sections omitting the primary antibody. Following incubation with biotinylated goat anti-Rabbit IgG (111–065-144, dilution 1:500, Jackson ImmunoResearch Laboratories, PA) for 60 min at RT, tissues were treated with ABC-HRP (Vector, PK-6100, Vector Laboratories, CA) and visualized using ImmPACT VIP Peroxidase Substrate (Vector, SK-4605). Finally, tissues were counterstained with 0.5% Methyl Green, dehydrated, cleared, and cover-slipped using DPX (Electron Microscopy Sciences, 13,512, Electron Microscopy Science, PA).

The following primary antibodies were used:
The following secondaries were used:
Following the standard immunohistochemistry protocol the brains were fixed again in 4% Paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) for four hours at room temperature. The brains were mounted on poly-L-lysine-coated cover slips and dehydrated through a series of ethanol baths (30%, 50%, 75%, 95%, and 3 × 100%) each for 10 min. Following dehydration they were submerged in 100% Xylene three times for 5 min each. Samples were embedded in DPX (DPX; Electron Microscopy Sciences, Hatfield, PA). Whole mount brain and VNCs were imaged using a Plan-Apochromat 20x/0.8 M27 objective (voxel size = 0.56 × 0.56×1.0 µm; 1024 × 1024 pixels per image plane). All images of brains expressing csChrimson::mVenus were visualised in 20x mode. Note that this protocol was also used to get extra, confirmatory images of split-GAL4 lines used in behaviour.

Light microscopy was performed on insect heads following the procedure described by Reisenman and collaborators (Reisenman et al., 2002 (link)). Briefly, freshly decapitated heads were fixed for 3 hr in a mixture of 2.5% glutaraldehyde and 2.0% paraformaldehyde in phosphate buffer (pH 7.3) with glucose and CaCl₂ added. After gradual dehydrated in 100% ethanol, they were embedded via propylene oxide in Durcupan ACM (Electron Microscopy Sciences no. 14040). We used glass knives mounted on a motorised microtome to serially section blocks at 2–5 µm. Sections were stained on a hot plate with Toluidine Blue-Basic Fuchsin and mounted on a slide with DPX (Electron Microscopy Sciences no. 13510). Photomicrographs were adjusted for brightness and contrast by using Adobe Photoshop CS2. A total of 10 individuals were prepared, sectioned, stained and analysed.