Jung multicut 2045 microtome

For immunofluorescence analysis, samples were fixed with 4% paraformaldehyde for 1 h [28 (link)], embedded in paraffin, and serial-sectioned (5 μm) with a Leica Jung Multicut 2045 Microtome (Leica). After deparaffinization and rehydration through a graded ethanol scale, sections were immersed in 10 mM sodium citrate buffer (pH 6.0) for 10 min in a microwave oven for antigen retrieval and then incubated for 30 min with a blocking solution (2% bovine serum albumin (BSA) and 0.1% Tween20 in phosphate-buffered saline (PBS), 138 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, 1.5 mM KH₂PO₄, pH 7.4). Sections were then incubated for 1 h at 37 °C [44 (link)] with primary antibodies (Alomone Labs, Jerusalem, Israel) all diluted 1:200 in blocking solution. The primary antibodies used are presented in Supplementary Table 1.
After washing with PBS, the specimens were incubated for 1 h at room temperature with goat anti-rabbit Cy3-conjugated antibodies (Abcam, Cambridge, UK, excitation 562 nm, emission 576 nm), diluted 1:250 in blocking solution. Nuclei were stained by incubating for 15 min with 49.6-diamidino-2-phenylindole (DAPI) 100 ng/ml in PBS (Sigma-Aldrich, Milan, Italy). Slides were mounted with CitiFluor (CitiFluor Ltd., UK) and examined with the Nikon Eclipse Ni fluorescence microscope (Nikon, Tokyo, Japan).

For histological analysis at light microscopy, samples were fixed with 4% paraformaldehyde overnight at 4 °C, embedded in paraffin, and serial-sectioned (5 μm) with a Leica Jung Multicut 2045 Microtome (Leica, Wien, Austria). After deparaffinization and rehydration through a graded ethanol scale, sections were stained with hematoxylin and eosin (H.E.) (Bio-Optica, Milan, Italy), for a general morphological view, with Masson Trichrome (M.T.) with aniline blue kit (Bio-Optica, Milan, Italy) to highlight the collagenic component of capsular connective tissue and silver impregnation (S.I.) histoenzymatic kit (Bio-Optica, Milan, Italy) to highlight argyrophilic neurofibrils in the capsular connective. Samples were observed with a Nikon Eclipse Ni light microscope (Nikon, Tokyo, Japan) and data were recorded with a DS-5M-L1 digital camera system (Nikon, Tokyo, Japan).

For glycogen detection, after dissection of the larva, the three regions of the midgut, with the enclosed intestinal content, were immediately fixed in 4% (w/v) paraformaldehyde in PBS for 2 h at room temperature and then overnight at 4 °C. After dehydration in increasing ethanol series, specimens were embedded in paraffin [72 (link)] and 7-µm-thick sections were obtained using a Jung Multicut 2045 microtome (Leica, Wetzlar, Germany). After deparaffinization, sections were stained with PAS kit (Bio-Optica, Milano, Italy), according to the manufacturer’s instructions, to detect the glycogen deposits in the midgut tissues, and then analyzed under Eclipse Ni-U microscope (Nikon, Tokyo, Japan) equipped with digital camera (Tucsen photonics, Fuzhou, China).
For the detection of ferric iron, whole mount staining of the entire midgut was performed. After isolation, the tissue was fixed in 4% (w/v) paraformaldehyde in PBS for 20 min, and then stained with Perls’ staining kit (Bio-Optica, Milano, Italy) according to the manufacturer’s instructions. Each region of the midgut was analyzed under NSZ-606 Zoom Stereo Microscope (Xiamen Phio Scientific Instruments, Xiamen, China) equipped with TrueChrome II S digital camera (Tucsen photonics, Fuzhou, China).