Axioplan2 upright microscope

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).

For assessment of the phosphorylation of MLKL and VZV antigen expression, uninfected and VZV-infected HT-29s were seeded onto coverslips and allowed to adhere overnight. Cells were then treated with T+S+V, or DMSO as a control, for 7–8 h. Cells were then washed in PBS and fixed with cytofix (BD). Cells were permeabilised with ice cold methanol for 10 mins, then washed in Tris buffered saline (TBS). Blocking was performed using 20% normal donkey serum (NDS, Sigma-Aldrich) then primary antibodies (diluted in 10% NDS) incubated overnight at 4 ^oC.
For detection of VZV antigens alone cells were fixed with cytofix (BD biosciences), washed in PBS and permeabilised with 0.1% Triton X-100 for 10 mins (Sigma-Aldrich). Blocking was performed using 20% normal donkey serum (NDS, Sigma-Aldrich) then primary anti-VZV antibodies (diluted in 10% NDS) incubated for 1 hr at room temperature. In both cases bound primary antibodies were detected using species-specific Alexa Fluor 488 or 594 conjugated secondary antibodies. Cells were then mounted in Prolong Gold antifade containing 4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI) (Life Technologies). Imaging was performed using a Zeiss Axioplan 2 upright microscope with an Axiocam camera (Carl Zeiss). Counting for pMLKL was performed by randomly imaging 10–20 non-overlapping regions of each slide and manually counting cells.

Stable MDCKII cells were polarized on glass coverslips for 7 days and induced with tetracycline for 24 hours before fixing with 4% paraformaldehyde for 15 minutes at RT. Cells were permeabilized with PBST (0.2% Tween 20 in PBS) for 20 minutes at RT and blocked with 10% serum (v/v in 0.05% Tween 20) for 30 minutes at RT. The primary antibody in 2% serum was added overnight at 4°C before the cells were washed three times in PBST and incubated in the secondary antibodies (diluted in 0.05% Tween 20) for 30 minutes at RT (antibodies listed in Supplementary Table S1). Cells were then washed three times in PBS followed by one wash in distilled water. Coverslips were air dried before mounting with Prolong Gold antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific, Altringham, UK). Cells were imaged on a Zeiss Axioplan2 upright microscope (Carl Zeiss, Oberkochen, Germany) using a 63× objective and captured using an Axiocam MrM camera (Carl Zeiss) through Aixovision v4.8.2 software (Carl Zeiss). Specific band pass filter sets for DAPI, FITC, and Texas red were used to prevent channel bleed through. Images were processed with Fiji¹⁶ (link).¹⁶ (link) For quantitative analysis, the membrane area was selected after subtracting background, and the average bestrophin 1 signal within the defined membrane area was calculated by the “ROI manager” tool in Fiji.

Phosphokinase analysis was performed using the Proteome Profiler Human Phospho-Kinase Array Kit ARY003 (R&D Systems)^{53 (link)}. Array images were analyzed using the GeneTools image analysis software (Syngene). For Immunofluorescence, breast cancer cells (5 × 103) were seeded on chambered slides, allowed to grow for 24 h and treated as indicated and subjected to immunofluorescence analysis^{34 (link)}. 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, CA, USA).

Fifteen BRCA2 mutant breast tumor sections (from Lombardi Cancer Center, Georgetown University, Washington DC) were used for BRE and CDC25A expression analysis. Slides containing tumor samples were deparaffinized, hydrated, and boiled in citrate buffer for 20 min for antigen exposure. After blocking, the slides were probed either with anti-BRE antibody (GTX20991; Genetex, 1:200 dilution) or with anti-CDC25A antibody (sc-97; Santa Cruz Biotech, 1:400 dilution). For control, one slide was stained with normal IgG (Santa Cruz Biotech). The signal was detected by Elite ABC HRP detection kit (Vectastain). Microscopic observation was performed using the Axioplan2 upright microscope (Zeiss). Intensities of staining were measured using Image J software. Staining intensities with values >0.45 (arbitrary unit) were considered as high expression and lower than 0.45 were considered as low expression.

β-galactosidase expression was detected by X-gal staining. Embryonic, neonatal or adult hearts were dissected in cold phosphate-buffered saline (PBS) and fixed in 2% paraformaldehyde (PFA), 0.2% glutaraldehyde in PBS at 4 °C for 30 min to 2 h, depending on age, followed by two washes in Rinse solution (2 mM MgCl₂, 0.2% Nonidet P40, 0.1% sodium deoxycholate in PBS). They were then incubated in Staining solution (Rinse solution containing 1 mg/ml 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside (X-gal), 5 mM K₄Fe(CN)₆ and 5 mM K₃Fe(CN)₆) at room temperature for 3 h to overnight, depending on staining intensity and age of sample. Imaging of whole hearts was performed using a stereo microscope (Leica M165C) equipped with a ProgRes CF Scan camera and ProgRes CapturePro software (Jenoptik).
For samples at embryonic day (E)13 and younger, following whole-mount imaging the X-gal-stained hearts were dehydrated through an ethanol series, cleared using Histo-Clear (National Diagnostics) and paraffin-embedded for sectioning. Sections measuring 10 μm were de-waxed, counter-stained using Nuclear Fast Red (Electron Microscopy Services) and imaged using an Axioplan 2 upright microscope (Zeiss) with a ProgRes C5 camera and ProgRes CapturePro software (Jenoptik).