Elyra confocal microscope

MTT assay was performed to assess cell viability. Expression levels of proteins of interest were analyzed through western blotting. Transwell migration and invasion experiments were performed to measure the metastatic potential of cells. Migration experiment was carried out as follows: cells were placed into the upper chamber (5 × 10⁴ cells per insert) with DMEM culture medium containing 0.1% FBS. Ten percent FBS was added to the lower well (6.5 mm diameter, 8 mm pore size; Costar, Cambridge, MA) as a chemoattractant. Cells were then treated with vehicle (DMSO, 0.5%) or inhibitors. After 24 h incubation, non-migrating cells were scraped from the upper surface of the membrane using a cotton swab. Cells remaining on the underside were fixed and stained with DAPI solution (1 mg/ml) and counted at 20x original magnification by inverted fluorescence microscopy. For invasion experiments, 8 mm filters were pre-coated with Matrigel (30 mg/filter) and incubated at 37 °C for 2 h, prior to following the protocol of migration assay. Three-dimensional culture models of normal and malignant mammary cells were established based on previously published protocols [31 (link)]. A Zeiss LSM 780 plus Elyra confocal microscope was used to visualize and capture the stained 3D acini.

Immunohistochemistry was performed to observe immune cells for the macrophage phagocytosis experiment. Cochleae were fixed with 10% buffered formalin and then decalcified using 10% EDTA at 4°C for 5 days. The cochleae were dissected in 10 mM PBS to collect whole-mount tissues containing the sensory epithelium, lower section of the spiral ligament, and osseous spiral lamina. Following dissection, whole-mount preparations were treated with 0.5% Triton X-100 to permeabilize the cells for 30 min at room temperature and then incubated in a blocking buffer (pH 7.4) for 1 h at room temperature. The tissues were subsequently incubated overnight at 4°C with an anti-mouse CD45 (goat polyclonal IgG, AF114, R&D System) at a concentration of 1:100 in 0.5% BSA and 0.25% Triton X-100 buffer. After incubation, the tissues were rinsed three times with 10 mM PBS and then incubated with a secondary antibody (Alexa Fluor 568 donkey anti-goat IgG, A11057, Thermo Fisher) at a concentration of 1:100 in 0.5% BSA for 2 h. The tissues were further stained with propidium iodide (50 μg/ml in 10 mM PBS) for 10 min. Following three rinses with 10 mM PBS, the tissues were mounted onto slides and examined using a Zeiss ELYRA confocal microscope.

The fat body was obtained from females treated with dsRNA (at least 3 females for each condition) on the 10th day after feeding, followed by fixation with 4% paraformaldehyde for 30 min. After fixation, the organ was incubated in 50 mM ammonium chloride for 1 h and washed in Tris-buffered saline (TBS) (50 mM Tris-HCl, pH 7.5, 150 mM NaCl). The samples were then incubated in a blocking solution of 3% BSA in TBS-T (TBS 0.1% Tween) for 2 h under slow agitation. After an additional wash with TBS, the sections were incubated with primary antibodies against KDEL (1:200; Abcam, cat# ab176333) overnight at 4°C. Then, 3 more TBS washes were performed for 5 min, and the samples were subsequently incubated with the secondary anti-rabbit IgG antibody Alexa Fluor 488 (1:500; Thermo Fisher cat# A32723) for 2 h at room temperature. Finally, the fat bodies were incubated for 15 min in 1 mg/mL Nile red and 2 mg/mL DAPI prepared in 75% glycerol. Tissues were placed on their respective slides submerged in N-propyl gallate and immediately photographed on a Zeiss Elyra confocal microscope in two independent experiments. The fat bodies were analyzed using a ×63 objective with ×2 magnification.