Vectorshield h 1000 mounting medium

Cason’s trichome staining was performed as described before [25 (link)], In brief, the heads of 5-day-old male flies were fixed in 4% paraformaldehyde buffer solution (pH, 7.4) at 4°C overnight, after which, paraffin-embedded preparations of the fly heads were sectioned at 10 μm thickness by using a HM 340E rotary microtome (Thermo Scientific Microm, Walldorf, Germany). Sections were dried at 40°C overnight and subsequently dewaxed with CitriSolv (Fisher Scientific, Fair Lawn, NJ) and rehydrated with a series of ethanol to phosphate-buffered saline solution. Rehydrated paraffin sections were soaked into Cason’s trichrome stain for 15 min, and slides were gently swashed in tap water with subsequent wash in distilled water three times. Excess of water was removed and samples were mounted with mounted with a Vectorshield H-1000 mounting medium (Vector Laboratories, Burlingame, CA).
For Congo red staining, sections were dewaxed and then stained in Congo red solution for 12 min, after which sections were rinsed in tap water and dehydrated in 50, 70% ethanol for 1 min, followed by the incubation in 100% ethanol for 4 min. Slides were dried and mounted with mounting medium. Images were observed and captured using EZ4 HD equipped with an Integrated 3.0 Mega-Pixel CMOS camera (Leica, Heerbrugg, Switzerland).

Cells were grown to 75% confluency on circular glass coverslips (VWR, Radnor, USA) in 24-well plates and either transfected or treated. For transfected cells, treatments were administered 18 h after transfection. For imaging, cells were fixed in 4% formaldehyde in PBS for 10 min at room temperature with agitation, followed by permeabilisation in 0.2% Triton X-100 in PBS for 7 min at room temperature with agitation. Coverslips were then blocked in 4% BSA in PBS at room temperature for 20 min with agitation. Primary antibodies were diluted 1:250 into 4% BSA in PBS and added to coverslips for 20 min at room temperature upside down on PARAFILM M (Sigma-Aldrich). After washing three times in PBS for 3 min each, secondary antibodies were added (1:500) in a solution of 4% BSA in PBS for 20 min at room temperature upside down on PARAFILM M in the dark. DAPI was added to the secondary antibody solution to stain nuclei (1:2000). Coverslips were then mounted on glass microscope slides (VWR) with Vectorshield H-1000 mounting medium (Vector laboratories). A Zeiss LSM710 and accompanying Zen software were used to acquire confocal images (Zeiss, Oberkochen, Germany). Images were processed for presentation using Photoshop. Antibodies used are listed in Supplementary Table 2.