Nis element image software

IHC was conducted as before [11 (link)]. Goat serum (5%, for BMPR1A, AMHR2, and FSHR) or horse serum (5%, for ACVR1) was used to block unspecific binding sites followed by an incubation with the primary antibodies listed in Table 1, overnight at 4 °C. Next respective biotinylated secondary antibodies (anti-rabbit or anti-goat IgGs; 1:300; 1.5 h; room temperature; Vector Laboratories, Burlingame, CA, USA) and avidin-biotinylated horseradish peroxidase complex (1:100; 40 min; room temperature; Dako, Glostrup, Denmark) were applied in succession. Finally, the antigen-antibody complex was detected upon incubation with 3,3′-diaminobenzidine (DAB, Sigma-Aldrich). Sections were counterstained with Hematoxylin QS (Vector Laboratories). Non-immune rabbit or goat IgG was used in place of primary antibody to ensure absence of non-specific staining. The stained sections were examined and photographed under a Nikon Eclipse Ni-U microscope using Nikon Digital DS-Fi1-U3 camera (Nikon, Tokyo, Japan) and Nikon NIS-ELEMENT Image Software.

A drop of hemolymph (50 μL) was allowed to settle for 10 min onto a glass slide, to permit hemocyte attachment. Afterwards, hemocytes were fixed by adding 100 μL of Bouin’s fluid for 30 min at room temperature (2 replicates of samples obtained from each of 6 snails). Then the slides were gently washed in PcABS and were stained with either Harris hematoxylin-eosin (HE stain). This fixation and staining procedure gave better results than the traditional Romanowsky-type stains, particularly for showing the eosinophily of the granules. Slides were mounted with Eukitt and a coverslip. Hemocyte morphology was examined and photographed under a Nikon Eclipse 80i Microscope using Nikon DS-Fi1-U3 camera and Nikon NIS-ELEMENT Image Software for image acquisition. Hemocyte counts were made with a Neubauer’s hemocytometer. Alternatively, living cells were observed using phase contrast microscopy, after 10 min attachment.

The histological processing involved sample fixation in dilute (1:2) Bouin’s fluid, dehydration in a graded ethanol series, clearing in xylene, paraffin-embedding (Histoplast, Argentina), and sectioning (5–10 μm-thick) on a rotary microtome (Microm). After staining with Gill’s hematoxylin-eosin, we examined and photographed the samples under a Nikon Eclipse 80i microscope using Nikon DS-Fi1-U3 camera and Nikon NIS-ELEMENT Image Software.
Also, we examined serially sectioned (10 μm-thick) Harris hematoxylin-eosin-stained lung samples under a Nikon Alphaphot-2 YS2 microscope equipped with a Nikon Digital Sight DS-5M camera. We selected the images of every fifth section for the 3D-reconstruction of the blood system and innervation of the lung. For this purpose, we used the software Reconstruct 1.1.0.0 (Fiala, 2005 (link)) and followed the procedure described by Ruthensteiner & Heß (2008) (link) to assemble the final PDF-model, as described in Rodriguez et al. (2019) (link).