Immunofix

Upon sacrifice, mice were decapitated, and whole heads were decorticated, fixed by immersion in Immunofix (Bio-Optica, Milan, Italy) for 24 h, followed by decalcification in Biodec R demineralizing solution (Bio-Optica), freshly replaced every 2 days, until adequate tissue softening was achieved (approximately 15 days). Then, the anterior part of the skull including the nasal cavities was dissected, embedded in paraffin, and cut transversely in 6 µm-thick sections, which were stained with hematoxylin and eosin (Feldman and Wolfe, 2014 (link)).

After the treatments with CZP-EUD-NPs, mice were sacrificed by decapitation. The head was quickly dissected and the anterior part of the snout including the nasal cavities was isolated. The specimens were fixed by immersion in Immunofix (Bio-Optica, Milan, Italy) for 24 h, followed by decalcification in Biodec R demineralizing solution (Bio-Optica), freshly replaced every 2 days, until adequate tissue softening was achieved (approximately 15 days). Then, the specimens were rinsed, dehydrated in graded ethanol and embedded in paraffin. Finally, 6 μm thick sections were cut, dewaxed and stained with haematoxylin and eosin (H&E). The effects of the different treatments on the nasal mucosa were observed in order to assess: (i) the integrity of the surface ciliated epithelium, (ii) the signs of mucus overproduction, (iii) the occurrence of vasodilatation and inflammatory infiltrate.

Cultured rabbit cornea samples were fixed in 4% paraformaldehyde (Immunofix^®, BIO-OPTICA Milano, Italy) in 0.1 M phosphate-buffered saline (PBS, pH 7.4) for 4 h at 4 °C. The samples were washed with a solution of phosphate buffer 0.1 M for 1 h, dehydrated in graded ethanol (30°–100°), cleared in xylene, and finally embedded in Paraplast^® (McCormick ScientificLLC, St. Louis, MO, USA). Serial sections (5-µm thick), were obtained by a rotary microtome (LEIKA 2065 Supercut) and were stained with the histological stain Hematoxylin-Eosin (H/E) method [32 (link)]. All samples were observed and photographed with an optical microscope Axioshop, Zeiss, equipped with a Sony^® DSC-85 camera.

The hnHDL‐immunized rabbits and the control animals were dissected 39 to 47 weeks after immunization, IFA injection or 0.15M NaCl injection, respectively. The rabbits were killed by an overdose of intravenous pentobarbital sodium and perfused with phosphate‐buffered saline (PBS) followed by Immunofix (Bio Optica Milano Spa, Italia) through the left ventricle. Aortic specimens were fixed for 24 hours in Immunofix and embedded in paraffin for light microscopy. Cross‐sections, 5‐μm thick, were stained with hematoxylin and eosin to visualize morphology as well as by the pentachrome method proposed by K. Doello for staining the extracellular matrix.¹⁰

Frozen tissue sections were fixed for 15 min with 4% paraformaldehyde (Immunofix, Bio-Optica, Milan, Italy; 05-K01015) in PBS, washed twice in PBS and permeabilized for 10 min with 0.5% Triton X-100 (Merck; X100) in PBS. After 1 h block with 1% bovine serum albumin (BSA, Merck; A3294) at room temperature, coverslips were incubated in a humidified chamber for 2 h at room temperature with a mouse anti-human HIF-1α (H1 alpha 67), (Novus Biologicals, LLC, Littleton CO, USA), rabbit anti-human NFκB p65, (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-human VEGFA ab46154, (Abcam, Cambridge, UK). Afterward, coverslips were washed with PBS 3 times (5 min/wash) and incubated for 1 h with goat anti-rabbit or goat anti-mouse IgG Alexa Fluor 555 fluorescent secondary antibody (1:200, Invitrogen, Carlsbad, CA, USA). Nuclei were stained with 100nM SYTOX™ green (Life Technologies, Thermo Fisher Scientific, Carlsbad, CA, USA). Finally, samples were washed with PBS 3 times (5 min/wash) and coverslips were mounted in ProLong Diamond Antifade Mountant (Life Technologies, Thermo Fisher Scientific, Carlsbad, CA, USA) and analyzed with an LSM510 confocal microscopy (Zeiss, Oberkochen, Germany). Quantification of the fluorescence intensity in the tubules and stroma of the control and FD patient was determined using Image J Software v1.51 (NIH, Bethesda, MD, USA).

Immediately after hemolymph sampling, the gills and digestive glands were quickly removed from ice and stored and fixed in immunofix (paraformaldehyde 4% in phosphate-buffered saline, Bio-Optica, Milan, Italy) for 12 h at room temperature for histopathological condition evaluation. An investigation under histological conditions of digestive glands and gills was performed. Sampled fractions of both tissues from each treatment group were collected in triplicate from three specimens. Tissues were embedded in paraffin and successively sectioned to 5-μm sections by using a rotative microtome (Leica, RM2235). The obtained sections were stained using hematoxylin and eosin for a qualitative histopathological examination using a light microscope (Leitz Diaplan, Germany). For detailed procedures, see Pagano et al. (2016) (link), Lauriano et al. (2019) (link), Zaccone et al. (2015) .