Dm 2500 led model microscope

Epidermis (Ep), proboscis (Pr), intestine (Int) and oocytes (Oo) were chosen as target organs in the onset of previous works on Eulalia viridis^{21 (link),22 (link)}.These works revealed specialised pigments cells in epidermis and proboscis, as well as scattered pigment granules in epithelial cells of the gut and ooplasm. In the present work, cryopreservation and cryotomy were enforced to evaluate the pigments’ native appearance, i.e., without interference of fixatives and other chemicals used in histological and cytological processing. For the purpose, E. viridis were snap-frozen in liquid nitrogen, sectioned and placed in optimal cutting temperature (OCT) medium in an appropriate tissue mould. The OCT medium containing the tissue was frozen to have a solid support which allowed to cut longitudinal sections with 5–15 μm thick in the cryostat at −20 °C in a cryomacrotome (CM3600 XP, Leica Biosystems). Sections were transferred to pre-adhesivated slides (Thermo Scientific Superfrost Ultra Plus) and stored at −80 °C until observation in a DM 2500 LED model microscope equipped with a MC 190 HD camera (both from Leica). Both brightfield and autofluorescence observations were made. Samples were marked with Hoechst 33258 fluorochrome for DNA counterstaining.

Samples of liver, spleen, and tumor were carefully excised fresh and immediately fixed in 10% (v/v) neutral-buffered formalin solution (Sigma-Aldrich) for 24 h. After fixation, the samples were processed as described previously. Liver and spleen sections were stained with Gill’s Alum Hematoxylin and counterstained with alcoholic Eosin Y (H&E). Tumors were stained with a tetrachrome stain (TC) for fibers and nuclei that include Alcian Blue, Weigert’s Iron Hematoxylin, and van Gieson’s dye [37 ], similarly to our previous work [6 (link)]. Sections of all samples were stained with neutral red to enhance contrast and thus detect metallic gold deposits with a purplish color. Finishing the staining, all the sections were dehydrated in ethanol, cleared in xylene, and mounted with DPX Mountant (Sigma-Aldrich). Histological analyses were made with a DM 2500 LED model microscope equipped with an MC 190 HD camera (both from Leica).