4 well chamber slide

Glioblastoma cells were seeded in 4-well chamber slides (Nunc) and stabilized overnight. The cells were fixed with 1% paraformaldehyde at 4 °C for 10 min and permeabilized with 0.1% Triton X-100 solution (cat. no. T8787, Sigma–Aldrich) at room temperature for 3 min. After treatment with 1% bovine serum albumin to minimize nonspecific binding, the cells were labeled with an anti-GFAP antibody, anti-human mitochondria antibody, anti-vimentin antibody, and anti-nestin antibody at 4 °C overnight and treated with the corresponding Alexa Fluor 488- or 594- conjugated IgG antibody (1:500; cat.no. A11008, A11029, A11032, A11037, A11055, and A21207, Life Technologies) for 1 h. Nuclear staining was performed using DAPI. Then, the slides were observed under a fluorescence microscope (Leica).

ARPE-19 cells (2.5 x 10⁴) were grown to 80% confluence in 4-well chamber slides (Nunc NALGENE, New York, USA) and serum starved overnight. Cells were treated with TGFβ1 (5 ng/ml) or co-treated with AqE (100 and 300 μg/ml) or AlE (50 and 500 μg/ml) or CA or CI (100 μM) for 48 h. DMSO (0.1%) was used as a vehicle control. After treatment, the cells were washed with PBS, fixed with 4% paraformaldehyde, permeabilized using 0.1% Triton-X100 for 10 min and blocked with 2% BSA for 60 min. After overnight incubation with primary antibody of αSMA (1:100) (Sigma-Aldrich) or vimentin (1:100) (Abcam) or ZO-1 (1:75) (Abcam), the chambers were rinsed with PBS, followed by incubation with appropriate secondary antibody (1:400) for 2 h. Unbound secondary antibody was removed by washing with PBST. The nucleus was stained with DAPI. The slides were air-dried and mounted using 1:1 mix of glycerol and PBS. All the images were captured using a fluorescence microscope (Carl-Zeiss). Experiments were repeated thrice and 5 random images were captured for a single chamber in the slide.

Breast cancer cells were treated with HNK for 24 h, and fixed with electron microscopy fixing buffer, rinsed, stained with 2% uranyl acetate (0.22 µm filtered, 1 h, dark) in 0.1 M maleate buffer and dehydrated through a graded series of ethanol (30–100%). Cells were embedded in EPON, sectioned, stained and examined with an H7600 transmission electron microscope (Hitachi, Tokyo, Japan)^{45 (link)}. Breast cancer cells (5 ×10⁵ cells/well) were plated in 4-well chamber slides (Nunc, Rochester, NY) followed by HNK treatment as indicated and subjected to immunofluorescence analysis⁷⁷ . Fixed and immunofluorescently stained cells were imaged using a Zeiss LSM510 Meta (Zeiss, Dublin, California, USA) laser scanning confocal system configured to a Zeiss Axioplan 2 upright microscope (Zeiss, Dublin, California, USA).

Cell death was investigated with the “in situ cell death detection” kit from Roche. Rat neonatal cardiomyocytes were cultured in 4-well chamber slides (Nunc, UK) at a density of 1.5 × 10⁵ cells per cm². Cells were washed twice with PBS and permeabilized for 1 hour at room temperature with 0.1% Triton (v/v) in 0.1% sodium citrate (v/v). They were then washed three times in PBS and incubated with the labelled enzyme for 1 hour at 37°C. They were washed twice, mounted in VECTASHIELD containing DAPI, and observed with an Olympus B40 microscope. Apoptosis was quantified by counting apoptotic cells in 25 different fields or 400 cells. Pictures were taken using a Media Cybernetics camera (Bethesda, MD, USA) and analysed with proimage plus software (Bethesda, MD, USA).