Permount medium

H&E staining of skin rafts was performed essentially as described previously [20 (link)]. Briefly, paraffin-embedded skin rafts were cut into 5-μm sections, mounted on Superfrost/Plus slides (Fisher Scientific, Pittsburgh, PA), deparaffinized, rehydrated in a series of ethanol dilutions and stained with Harris hematoxylin (Fisher Scientific) and Eosin Y (Protocol, Fisher Scientific). Sections were mounted in Permount medium (Fisher Scientific) and analyzed at 10x and 20x magnification using AxioImager A2 microscope equipped with an AxioCam camera and AxioVision image capture software (Carl Zeiss MicroImaging, Inc., Thornwood, NY).

Liver samples were collected and fixed in 10% neutral buffered formalin (NBF). After dehydration using gradient ethanol, the samples were processed twice using xylene and embedded in paraffin. Tissue sections (6 μm in thickness) were made, spread on a slide and baked at 37 °C for 2 h. The slides were stained with H&E, mounted with Permount medium (Fisher Scientific), and examined under an optical microscope (ECLIPSE Ti, Nikon). Image quantification was carried out using NIS-Elements imaging platform from Nikon Instruments Inc. (Melville, NY). For Oil red O and Nile red staining, frozen liver samples were cut at 8 μm in thicknesses and fixed using 10% NBF. Liver triglyceride content was determined following a previously reported method with some modification^{46 (link)}. In brief, freshly collected liver tissues (100–200 mg) were homogenized in a mixture of chloroform and methanol (2:1) and tissue homogenates incubated overnight at 4 °C. The mixture was centrifuged at 12,000 rpm for 20 min and supernatant collected. The collected fractions were dried and lipids re-dissolved in 1% Triton X-100. The triglyceride concentration was determined using a commercial kit (#TR22203) from Thermo-Scientific (Waltham, MA).

Wing-specific GAL4 drivers were grown at 25°C. Virgins were collected twice a day from the bottles. Virgins were crossed with males that carry the indicated UAS-TRiP line constructs in a ratio of female:male of 10:3. Adult flies were harvested within 7 days of eclosure. Wings were removed and mounted on microscopy slides to obtain high resolution images. 360 wings were analyzed in different case studies for this paper. Wings were placed in ethanol, and approximately 15 wings were mounted on each slide in Permount medium (Fisher Scientific, SP15) using standard procedures. For the benchmark experiments related to InsR, slides were batch-imaged using an EVOS microscope at ×4 magnification. For the case study involving wings from four Drosophila species, the wings were incubated overnight in 70% ethanol. Wings of different Drosophila species were imaged using a Nikon Eclipse Ci-S microscope using a Jenoptik ProgRes® monochromatic camera and the ProgRes® Capture Pro 2.9 software.

Frozen sections were air dried at room temperature for 10 min. Sections were stained in hematoxylin solution (Surgipath 01522) for 1 min. Sections were then washed with ultrapure water or Milli-Q® (MQ) water for 4 min (twice), acidic alcohol solutions for 5 min, and MQ water for 4 min. Sections were then immersed in ammonia water (or sodium bicarbonate) 5–6 times slowly, MQ water for 4 min, and graded ethanol (EtOH) in the following order: 80% EtOH for 4 min, 95% EtOH for 1 min (three times), 100% EtOH for 1 min, 100% EtOH for 3 min, Xylene for 5 min, and mounted with Permount medium (Fisher Scientific).

After decapitation, the right brain hemispheres were rapidly removed, post fixed in cold 4% paraformaldehyde, and cryoprotected in sucrose solution. The brains were then cut on a freezing microtome into 30 μm frontal sections (AP −4.4 to 6.6 mm from bregma according to [54 ] for SNc–ventral tegmental area (VTA)) according to the stereological rules and stained as described before [57 (link)]. Free-floating sections were incubated in primary antibodies (anti-tyrosine hydroxylase (TH; AB_2201526); anti-GFAP (AB_2109645), both from Chemicon Int., USA; anti-Iba1 (AB_839504; WAKO, Japan)). For anti-S100 (AB_306716; Abcam, UK) staining heat-induced antigen retrieval in 10 mM citrate buffer pH 6.0 was performed. After incubation with secondary antibodies (anti-mouse (AB_2313571) or anti-rabbit (AB_2313606) biotinylated, Vector Laboratories, UK), sections were processed using an ABC-Peroxidase Kit (Vector Laboratories, UK) and 3,3′-diaminobenzidine as a chromogen. Subsequently, sections containing SNc–VTA stained for TH were counterstained with 1% cresyl violet (CV) with Nissl method. All sections were cover-slipped in a Permount medium (Fisher Scientific, USA).

Quantification of atrial interstitial fibrosis was performed using the Picrosirius red staining technique, as previously described [59 (link)]. The left atria were removed from the mouse hearts under anesthesia with 2% isoflurane, and fixed overnight with 10% buffered formalin at 4 °C. The atria were embedded in paraffin and cut to a thickness of 8 µm using a microtome. Sections were deparaffined using xylene and rehydrated with an ethanol-water gradient. Sections were then stained for 1 h with a saturated picric acid solution containing 0.1% Direct Red 80 (Sigma-Aldrich Corp., St. Louis, MO, USA), and washed twice with 0.5% acetic acid solution. The slides were dehydrated with a water-ethanol gradient before cover slipping them with Permount medium (Fisher Scientific, St-Laurent, Qc, Canada). Images were acquired using Qicam optic camera (Qimaging Corp., Tucson, AZ, USA) mounted on a brightfield microscope and analyzed using ImageJ software, version 1.8.0 (National Institutes of Health [NIH], Bethesda, MD, USA).