Cason's Trichrome and Congo Red Staining of Fly Heads

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).