Alcian Blue

Since the larval exoskeleton is resistant to known fixative reagents, we performed an adequate fixation by injecting buffered formalin 10% with an insulin syringe in the last left proleg. The volume of solution injected, to have a turgid consistency of the larvae, was about 100 µL. Larvae were then stored at 4°C for 24 h, to fix internal organs and block melanization. Whole larvae were dissected transversally or sagittally into two halves by means of an anatomic pincers and by using a new lancet blade for each larva. The procedure was carefully performed to avoid the squeeze of the larval tissues. The two halves of larvae were placed in the same BioCassette and routinely processed in the path lab. For transversally sectioned larvae, each paraffin-embedded half was further sectioned into two/three rings, after cooling the larva at room temperature for a few minutes to harden the tissues. A crucial step to avoid the paraffin block rupture during microtome sectioning was to keep the cut larval tissues in hot paraffin for one hour to stabilize the inclusion, and to obtain a complete merge of the cut rings in the final paraffin block. Finally, four/six rings (one in the distal part, two in the middle, and one in the proximal part) were positioned in each paraffin-block. Histochemical staining on slides with serial tissue sections was then performed: haematoxylin and eosin (HE) was used to evaluate tissue morphology, periodic acid Schiff (PAS) and Grocott Methenamine staining (GMS) to highlight fungi localization and host interaction, Giemsa, Alcian blue at various pH (1, 2.5, and 3.1) to evaluate hemocytes, and Feulgen staining to evaluate DNA. All histo-chemical stainings were performed according to standard laboratory protocols.¹⁴ Sagittally sectioned larvae were routinely paraffin-embedded.¹³ A distance of 50 µm was maintained between serial 4-micron-thick tissue sections of the two halves, and the slides were stained with haematoxylin and eosin.
The microscopic visualization has been performed using a Leica Microscope DMLB, and the image acquisition with the NanoZoomer-XR C12000 series (Hamamatsu Photonics K.K., Tokio, Japan.).

Animals were euthanized with an overdose of sodium pentobarbital (100 mg/kg, intraperitoneally) after in vivo hemodynamic measurement, and then were rapidly perfused via inferior vena cava with precooled physiological saline for 5 min. The hearts were quickly removed, sectioned, and prepared for paraffin embedding and cryo-section. Routine staining techniques in paraffin embedded tissue (7-µm) included hematoxylin and eosin (H&E) staining (25˚C, 15 min) and Masson's trichrome staining for evaluating interstitial collagen deposition. Tetraethyl rhodamine isothiocyanate-conjugated wheat germ agglutinin (Invitrogen; Thermo Fisher Scientific, Inc.) plus 4,6-diamidino-2-phenylindole (DAPI; 5 mg/ml; Vector Laboratories, Inc.) staining was used to measure cardiomyocyte cross-sectional area, which was examined with a fluorescent microscope (80i; Nikon Corporation) as previously described by the authors (13 (link)). All image analysis was performed in a blinded manner by Image Pro Plus (version 4.5; Media Cybernetics, Inc.).
For detecting lipid droplet, Oil Red O staining was used in cryo-sectioned heart tissue (10 µm), and the sections were fixed with 10% buffered formalin (25˚C, 24 h) and stained with Oil Red O working solution (25˚C, 30 min). Hematoxylin was used for counterstaining. The perirenal adipose tissue was removed and smeared over a slide serving as the positive controls. For detecting sulfated proteoglycans, paraffin-embedded sections were stained with toluidine blue. Alcian blue/periodic acid-Schiff (AB-PAS) was used in paraffin-embedded sections to detect acidic sulfated mucins (AB positive), O-glycosides (PAS positive) and sialic acid (PAS positive) as previously described (14 (link)).
Transmission electron microscopy was performed for evaluating myocardial ultrastructure. Briefly, the samples from LV were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2; 4˚C, 24 h), post-fixed in 1.0% OsO4, dehydrated in alcohol and acetone solution, embedded in Epon812, sectioned with LKB ultramicrotome, and stained with uranyl acetate followed by lead citrate, then observed with a transmission electron microscope.

Mice were sacrificed at the end of the experiment. Parts of the colons were fixed in 4% paraformaldehyde cleared in xylene, embedded in paraffin, and cut into 5 mm thick slices. The histopathological performance of colon tissue was stained with hematoxylin-eosin (HE). Furthermore, the mucous cells in the colon were stained with Alcian blue/periodic acid-Schiff (AB/PAS). All experiment processes were performed according to the manufacturer's instructions.