Zen 2.6 lite

Nectar guide tissues at the same site near the throat (marked by a red circle in Fig. 1a) of the corolla were cut into small pieces. The trichomes in these tissues were then imaged under a light microscope, and their lengths were measured using Zeiss ZEN 2.6 lite (blue edition) software.
Fig. 1

Phenotypes of nectar guide trichomes. (a) Front view of the whole corolla (upper) and enlarged view of the corolla throat of M. lewisii (Mlew, left), M. parishii (Mpar, middle), and their F1 hybrid (right). Scale bars, 5 mm (top row) and 1 mm (bottom row). The red circle marks the position where nectar guide trichome length were quantified. (b) Front view of F2 hybrids representing the four classes. Scale bars, 4 mm. (c) Light microscopy images of nectar guide trichomes of M. lewisii, M. parishii and F1 hybrids. Scale bars, 200 μm. (d) Quantification of nectar guide trichome length (n = 20 for each genotype). Error bars are 1 SD. Asterisks indicate differences from M. lewisii (** p < 0.01, student’s t test). (e) Frequency distribution of the four classes of F2 individuals

Pharmacological effects of the peptides and saline solution on DAD were assessed on days 7, 14 and 30.
After the animals were euthanized, the lungs were extracted and filled with 10% neutral formalin solution. The tissue specimens were rinsed in running water, dehydrated in an ascending alcohol series and embedded in paraffin. Then, 4–5 µm paraffin sections were stained with hematoxylin and eosin and examined by ordinary light microscopy using an AxioScope A1 (Carl Zeiss, Oberkochen, Germany). Microphotographs of the histological sections were made with Axiocam 305 color high-speed camera (Carl Zeiss, Oberkochen, Germany) and the software ZEN 2.6 lite (Carl Zeiss, Oberkochen, Germany). Histological examination included the assessment of the following morphological characteristics: peribronchial and perivascular infiltration with the mononuclear cells, infiltration of alveolar walls and ducts with the mononuclear cells, sites of pulmonary collapse and the presence/absence of necrotic foci.
The extent of different inflammatory manifestations in the lungs was evaluated using a semiquantitative scoring scale: 0—none (within the normal range); 1—minimal; 2—mild; 3—moderate; 4—severe, tissue alterations are noticeable, but there is a potential for increase in severity; 5—very severe, the maximal extent of alterations, characterizing the total lobe injury.

To determine the expression of galectin‐9 in HaCaT and cervical cell lines, we performed an immunocytochemistry (ICC) and immunofluorescence assay (IF). A protocol suggested by Abcam for ICC and IF was followed. HaCaT, SiHa and HeLa cell lines were cultured in an eight‐well Chamber Slide™ system (C7182; Merck) and fixed with 100% methanol for 5 min at room temperature. Nonspecific antibody binding was blocked with 1% BSA in PBS for 1 h. Then cells were incubated overnight at 4 °C with an anti‐galectin‐9 antibody (ab69639; Abcam) diluted 1 : 400 with 1% BSA in PBS. Next, cells were incubated for 1 h at room temperature with anti‐rabbit IgG (Alexa Fluor® 488) (ab150077; Abcam) diluted 1 : 1000 in 1% BSA in PBS. Nuclei staining was performed with 0.1 µg·mL⁻¹ DAPI Stain (4083; Cell Signaling Technology, Danvers, MA, USA). An isotype IgG control was applied as a negative control test. Fluorescence intensity was determined using the program zen 2.6 Lite from Zeiss (Oberkochen, Germany). The ratios of cytoplasmic and nuclear level fluorescence were also determined for each cell line.