Polysine coated glass slides

Pellets from CT26 parental and hyperploid clones were fixed in formalin for 4 h at RT and then embedded in paraffin. Sections of 4 μm were obtained by means of a RM2245 microtome (Leica Microsystems GmbH, Wetzlar, Germany) and then applied onto histological Polysine®-coated glass slides (Thermo Fisher Scientific). Then samples were deparaffinized in xylene and rehydrated by incubation following 95%, 70%, 50%, 30%, (v/v in PBS) ethanol baths (2 min/bath). HES staining was performed following standard procedures.

Routinely, the G. mellonella larval tissues were fixed in phosphate‐buffered 4% paraformaldehyde for 7 d, dehydrated, and embedded in Paraplast (Sigma‐Aldrich, St. Louis, Mo, USA). Next, they were sectioned with a rotating microtome to obtain 5 µm thick Paraplast sections, which were mounted on polysine‐coated glass slides (Thermo Fisher Scientific, Braunschweig, Germany). Next, the larval sections were deparaffinized in xylene (2 times for 5 min), rehydrated by passing through decreasing concentrations of alcohols (100%, 95%, and 70% for 5, 3, and 3 min, respectively), and stained with hematoxylin (Sigma‐Aldrich) for 7 min. The sections were then rinsed for 20 min with running tap water and deionized water for 5 min. After that step, the sections were stained with eosin (15 min, Sigma‐Aldrich) and rinsed again. Next, the slides were dehydrated for 3 min in each alcohol solution (70%, 95%, 100%) and embedded in xylene dibutyl phthalate (DPX, Sigma‐Aldrich). Two solutions were used for tissue staining with calcofluor white: solution A (containing 9% KOH and 9% glycerol) and solution B (containing 0.1% calcofluor white, Fluka, Switzerland). Immediately before staining, solutions A and B were mixed in a 1: 1 ratio and the larval sections were immersed in the mixture for 10 min in the dark. All specimens were analyzed using a Carl Zeiss Axiovert 200M confocal microscope (Germany).

For analysis by real-time PCR, ELISA and western blot, enucleated eyes were dissected immediately on chilled PBS and processed as described previously^{60 (link)}. For immunohistochemistry, enucleated eyes were first fixed in 4% paraformaldehyde (PFA) in 1× PBS. Eyes were treated to sucrose gradient (15% and 30%) cryoprotection before embedding in Tissue Freezing Medium® (Leica Biosystems Inc., Buffalo Grove, IL, USA) on dry ice. Retina sections of 6–8 µm thickness were cut using HM525 HX Cryostat (Thermo Fisher Scientific, Waltham, MA, USA) and collected on Polysine™-coated glass slides (Thermo Fisher Scientific). Glass slides were kept frozen at −80 °C until staining was performed. For flat mount staining, eyes fixed in 4% PFA for 10 min was hemisected ora ciliaris to isolate the neural retina. Flat mount staining was performed immediately.