Eclipse light microscope

Spheroids of breast cancer cells were developed and cultured as previously described [13 (link)] using a serum-free mammary epithelium basal medium (Gibco, USA). After 7 days of culture, the numbers and morphology of spheroids were observed using a Nikon Eclipse light microscope. Then, the MDA-MB-231 cells-derived spheroids were treated with citral at different concentrations (2.5, 5.0 and 10.0 μg/mL) for 48 h. After the treatment period, vehicle control and citral treated spheroids were observed under Nikon Eclipse light microscope (the image of 4 random areas per well were taken for each group, and the experiment was carried out in triplicate) and the average volume of the spheroid was calculated using V = (4/3) π R³. (n = 12 images per group). The harvested spheroids were also dissociated into single cells by incubating the spheroids in enzymatic solution using 0.25% trypsin-EDTA (Gibco USA) for 30 min followed by mechanical dissociation using pipetting. The harvested single cells suspension were subjected to the following assays.

The remaining six mice in each group were used for histopathological examination. Their lungs were fixed with zinc fixative (BD Biosciences, Franklin Lakes, NJ, USA). After separation of the lobes, 2-mm-thick blocks were taken for paraffin embedding. The embedded blocks were sectioned (thickness 3 μm), and the slices were stained with alcian blue (AB) to evaluate the degree of mucus secretion in the bronchial epithelium (from proximal to distal parts). To determine the number of eosinophils in the submucosa of the airway, the slices were also stained with a slightly modified original luna stain [26 ]. The slices were immersed in a hematoxylin solution and then in Biebrich scarlet solution for 15 and 20 min, respectively. After a rinse with tap water, the slices were dipped in 1 % hydrochloric acid (in ethanol) six times, followed by five dips in a 0.5 % lithium carbonate solution. Five randomly selected visual fields were photographed by means of a light microscope (400× magnification). The number of eosinophils was determined. Histopathological examination of inflammatory cells and epithelial cells in the airway was performed under a Nikon ECLIPSE light microscope (Nikon Co., Tokyo, Japan).

All mice were used for pathological examination. The lungs were fixed by 10% neutral phosphate-buffered formalin. After separation of the lobes, 2-mm-thick blocks were taken for paraffin embedding. Embedded blocks were sectioned at a thickness of 3 µm, and then stained with May-Grunwald’s stain solution (Nacalai tesque, Inc, Kyoto, Japan) and Giemsa’s azur eosine methylene blue solution (Merck KGaA, Darmstadt, Germany) to evaluate the degree of infiltration of eosinophils and lymphocytes in the airway from proximal to distal. The sections were stained with periodic acid-Schiff (PAS) to evaluate the degree of proliferation of goblet cells in the bronchial epithelium. A pathological analysis of inflammatory cells and epithelial cells in the airway was performed using a Nikon ECLIPSE light microscope (Nikon Co., Tokyo, Japan). The degree of infiltration of eosinophils and lymphocytes in the airway or proliferation of goblet cells in the bronchial epithelium was graded in a blinded fashion: 0, not present; 1, slight; 2, mild; 3, moderate; 4, moderate to marked; 5, marked. ‘Slight’ was defined as less than 20% of the airway with eosinophilic inflammatory reaction or with goblet cells stained with PAS; ‘mild’ as 21–40%; ‘moderate’ as 41–60%; ‘moderate to marked’ as 61–80%; and marked as more than 80% of the airway [8 (link), 16 (link)].