Axio imager z1 fluorescence microscope

Cells were washed twice in PBS, then fixed in freshly prepared 3.7% paraformaldehyde in PBS (pH 7.4) for 30 min at 4 °C, washed in PBS for one time and permeabilized in 0.1% Triton X-100 in blocking solution (3% goat serum plus 0.1% BSA in PBS) for 30 min at room temperature, then washed in PBS for one time, and left in blocking solution for 2 h. Cells were incubated overnight at 4 °C with primary antibodies against Oct4 (sc5279; Santa Cruz), Nanog (ab80892; Abcam), SSEA-1 (MAB4301; Millipore), βIII-tubulin (CBL412; Chemicon), alpha 1-fetoprotein (AFP; DAK-N1501; Dako), alpha smooth muscle actin (α-SMA; ab5694-100; Abcam), γH2AX (05-636; Millipore), TRF1 (TRF12-S; Alpha Diagnostic) and Zscan4 (AB4340; Millipore). Then cells were washed three times (each for 15 min) with blocking solution, and incubated for 2 h with secondary antibodies at room temperature. Goat Anti-Mouse IgG (H + L) FITC (115-095-003; Jackson) and Goat Anti-Rabbit IgG (H + L) Alexa Fluor^® 594 (111-585-003; Jackson), diluted 1:200 with blocking solution, were used. Samples were washed, and counterstained with 0.5 μg/ml Hoechst 33342 (H1398; MP) in Vectashield mounting medium. Fluorescence was detected and imaged using a Zeiss Axio-Imager Z1 fluorescence microscope.

Cells grown on glass coverslips were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 25 min, followed by permeabilization in 0.5% Triton X-100 in PBS for 8 min, or fixed with methanol at −20 °C for 3 min. Cells were blocked with 2% bovine serine albumin in PBS and incubated with primary antibodies and then FITC- or rhodamine-conjugated secondary antibodies, followed by staining with DAPI as described previously^{43 (link)}. For visualization of actin filaments, cells were stained with FITC-conjugated phalloidin for 30 min. Coverslips were mounted and examined with an Axio Imager Z1 fluorescence microscope (Carl Zeiss).

Confluent HFF cells were grown on glass coverslips and infected with T. gondii. After 18 to 36 h, the coverslips were fixed with 3.7% formaldehyde in phosphate-buffered saline (PBS) and processed for immunofluorescence as described (62 (link)). Primary antibodies were detected by species-specific secondary antibodies conjugated to Alexa Fluor 488/594 (ThermoFisher). Coverslips were mounted in Vectashield (Vector Labs, Burlingame, CA), viewed with an Axio Imager.Z1 fluorescence microscope (Zeiss), and processed with ZEN 2.3 software (Zeiss).
For Western blot, parasites were lysed in 1× Laemmli sample buffer with 100 mM DTT and boiled at 100°C for 10 min. Lysates were resolved by SDS-PAGE and transferred to nitrocellulose membranes, and proteins were detected with the appropriate primary antibody and corresponding secondary antibody conjugated to horseradish peroxidase. Chemiluminescence was induced using the SuperSignal West Pico substrate (Pierce) and imaged on a ChemiDoc XRS+ (Bio-Rad, Hercules, CA).

For PAS staining, samples of isolated retinal microvasculature were mounted differently after elastase digestion and clearing. Specifically, the isolated retinal microvasculature was dried overnight after being mounted on glass Superfrost Plus microscope slides (Fisher Scientific, #12-550-15). On the following day, the samples were first rehydrated in distilled water for 15 min. The rehydrated samples were then incubated with periodic acid (MilliporeSigma, #P7875) solution at a concentration of 35 mM at RT for 8 min, followed by a brief dipping in distilled water. Afterward, the tissues were stained with Schiff (Sigma-Aldrich, #3952016) for 15 min, followed by three separate extensive washes in running distilled water lasting 5 min each time. The tissues were then stained with hematoxylin (Richard-Allan Scientific, #7231) for 2 min, followed by three 5-minute distilled water washes. After staining, the slides were dehydrated in 70%, 85%, 90% and 100% ethanol, and finally xylene, 2 min for each reagent. Following this, the slides were mounted with Permount mounting medium (Fisher Scientific, #SP15-100). For purposes of quantification, the images were obtained using an Axio Imager Z1 fluorescence microscope (Carl Zeiss MicroImaging, Inc.) equipped with ApoTome, AxioCam MRm, and AxioCam HRc cameras (for more details see Stereological quantification below).

Hela cells were seeded on coverslips (10 mm×10 mm) in a 24-well plate. After 24 h, cells were transfected with 2μg indicated expression plasmids (1 μg/each expression vector) by Lipofectamine 3000 (Thermo) according to the manufacturer’s instructions. After 24 h since transfection, cells we washed twice in PBS, fixed in 4% paraformaldehyde solution, and permeabilized by 0.1% Triton X-100 in PBST (0.05%Tween-20 in PBS). After washing with PBST three times, the wells were blocked with PBST containing 10% (wt/vol) BSA at room temperature for 1 h. Cells were further incubated with primary mAb (Sigma) at 4°C overnight, washed three times in PBST, and incubated with the second antibody Alexa Fluor 568 Goat anti-rabbit IgG (Invitrogene) or Alexa Fluor 488 Goat anti-mouse IgG (Invitrogene) for 1 h. Following triple washing in PBS, cells were stained with 0.2 μg/mL DAPI for 5min. The slips were washed three times in PBS, mounted in ProLong™ Glass Antifade Mountant (Thermo) and photographed with a Carl Zeiss Axio Imager Z1 fluorescence microscope.

Cells were fixed with 4% paraformaldehyde 4d pi and washed with PBS, permeabilized with 0.2% Triton-X in PBS (0.2% PBST) and blocked with 5% donkey serum in 0.2% PBST. The cells were stained overnight at 4°C with anti-HNF4α (Ab41898, Abcam), anti-Flavivirus Group Antigen (clone D1-4G2-4-1, Millipore), anti-ZIKV NS3 (kindly provided by Andres Merits, Institute of Technology, University of Tartu, Estonia), anti-albumin (ALB; A0001, Dako), anti-α-fetoprotein (AFP; A008, Dako), anti-sodium taurocholate cotransporting peptide (NTCP; HPA042727, Sigma-Aldrich) and anti-Caspase 3 (active, cleaved form) (AB3623, Millipore) antibodies. Afterwards, the cells were incubated for 30 min at room temperature with the appropriate secondary antibodies and Hoechst (Sigma-Aldrich). Images were taken using the AxioimagerZ.1 fluorescence microscope (Carl Zeiss Inc.). Appropriate isotype control antibodies were used in all immunofluorescence staining experiments.