Observer a1 inverted microscope

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was used for detecting DNA fragmentation in frozen kidney tissue sections (3 μm) by using In Situ Cell Death Detection Kit, Fluorescein (Roche, USA). Briefly, the frozen tissue slides were fixed by 4% fresh-prepared paraformaldehyde solution for half an hour at room temperature at beginning and then washed by PBS buffer for another thirty minutes. The slides were further incubated with ice cold permeabilisation buffer (freshly prepared 0.1% Triton X-100, 0.1% sodium citrate) for two minutes and washed completely by PBS. For the last step, the tissue in the slides was covered by 50 μL TUNEL reaction buffer with fluorescein at 4°C overnight. On the following day, the slides were washed thrice in PBS and mounted by DAPI containing gum. The pictures were observed and evaluated under fluorescence microscopy (Observer A1 inverted microscope, ZEISS, Germany) with 200x actual magnification. Excitation wavelength in the range of 450 nm to 500 nm and detection wavelength range of 515–565 nm were used for TUNEL fluorescence and the DAPI staining was observed with an excitation wavelength of 340 nm. The cells that were positively labeled were quantified in ten different fields per slide by Image pro plus 6.0 software (Media Cybernetics, Georgia, USA).

Immunofluorescence staining was performed for macrophage biomarker F4/80 and neutrophil membranous biomarker Ly6G. Removed kidney tissues were frozen immediately with liquid nitrogen and then were cut into 2 μm thick frozen sections with the help of freezing microtome (Leica Biosystems, Germany) by skilled technician. Endogenous peroxidase was blocked for 20 min in 3% hydrogen peroxide, and PBS was used to rinse the samples. After blocking with 5% BSA, the slides were incubated at 4°C with a rabbit anti-mouse Ly6G (dilution 1 : 100; Abcam, USA) and F4/80 antibody (1 : 50, Invitrogen, Life Technologies, USA) overnight. The anti-rabbit fluorescein-conjugate second antibody (Invitrogen, Thermo Fisher Scientific, USA) was used to detect and amplify the primary signals by light emission. The pictures were observed and evaluated under fluorescence microscopy (Observer A1 inverted microscope, ZEISS, Germany) with 400x actual magnification. Two observers evaluated the slides, who were unaware of the slides classification. F4/80-positive and Ly6G-positive cell number in each section were calculated by counting positively stained cells in 10 fields per slide at 400x magnification.

Briefly, fluorescence microscopy was done on an Observer A1 inverted microscope (Zeiss, Germany) using a 25 × 0.8 numerical aperture water immersion objective and a 175 W Xenon lamp as a light source. Before imaging, DRG neurons were incubated with 4 μM Fura-2 at 37 °C for 30–40 min and washed with extracellular solution at 37 °C for another 30 min. Excitation light was passed either through a 340-BP 30 filter or a 387-BP 16 filter. Two filters were switched by an ultra-high speed wavelength switcher Lambda DG-4 (Sutter, Novato, CA). Emissions elicited from both excitation wavelengths were passed through a 510-BP 90 filter and collected by a charge-coupled device camera (Zeiss). Different solutions were applied by multi barrel perfusion system (WAS02, DITEL, Prague). AxioVision software (Zeiss) was used to record image data. After background subtraction in each channel (B₃₄₀, B₃₈₀), the ratio (R) of fluorescence elicited by two excitation light, B₃₄₀ and B₃₈₀, was calculated: 59 (link). Data weres analysed by using AxioVision and Matlab (MathWorks, Natick, Massachusetts).

Calcium imaging was performed as previously described (Chen et al., 2014 (link)). Fluorescence microscopy was done on an Observer A1 inverted microscope (Zeiss, Germany) using a 25 × 0.8 numerical aperture water immersion objective and a 175W Xenon lamp as a light source. Before imaging, DRG neurons were incubated with 4 mM Fura-2 at 37°C for 40 min and washed with extracellular solution at 37°C for another 30 min. Excitation light was passed either through a 340 BP 30 filter or a 387 BP 16 filter. Two filters were switched by an ultra-high speed wavelength switcher Lambda DG-4 (Sutter, Novato, CA). Emissions elicited from both excitation wavelengths were passed through a 510 BP 90 filter and collected by a charge-coupled device camera (Zeiss). Different solutions were applied by multi barrel perfusion system (WAS02, DITEL, Prague). AxioVision software (Zeiss) was used to record image data. After background (B₃₄₀, B₃₈₀) subtraction in each channel (F₃₄₀, F₃₈₀), the ratio (R) of fluorescence elicited by two excitation light was calculated: R=(F₃₄₀ – B₃₄₀) / (F₃₈₀ – B₃₈₀). Data was analyzed in AxioVision, Matlab (MathWorks, Natick, Massachusetts), and GraphPad prism (GraphPad Software Inc., San Diego, CA).

MCF-7, T47D, MDA-MB-231, and Slug overexpressing or knockdown cell lines MCF-7Slug, T47DSlug and MDA-MB-231shSlug were cultured in Millicell EZ SLIDE 8-well glass (Merck Millipore, Darmstadt, Germany) to 80% confluence. After removing the medium and washing once with PBS, cells were fixed via 4% paraformaldehyde. Then cell membranes were permeated with 0.5% Triton-X-100 for 5–8 min. Heterogenetic antigens were blocked with 2.5% goat serum for 30 min, then cells were incubated with anti-Slug and anti-ERα antibody diluted 1:400 and 1:200, respectively, overnight at 4 °C. In the next day, after incubation with secondary antibody, cells were mounted in DAPI (Invitrogen Corp.) for 3–5 min. Slides were analyzed using a ZEISS Observer A1 inverted microscope (× 400 magnification) (Carl Zeiss).