Zen digital imaging for lsm 700

Cells were plated on poly-d-lysine (Sigma)-coated microscope cover glasses (Thermo Fisher Scientific). For immunocytochemistry, cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences) for 15 min and immersed in guanidinium thiocyanate for epitope retrieval. Then the cells were blocked with 1% bovine serum albumin in PBS with 0.1% Tween-20 for 30 min and probed with the first antibodies at 4 °C overnight: anti-PrP monoclonal antibody, Sha31 (Spi-Bio), and anti-β-tubulin polyclonal antibody (Novus). To visualize the target molecules, cells were then incubated with Alexa Fluor 594-conjugated or 488-conjugated secondary antibodies (Invitrogen). Counterstaining for nuclei was performed with Hoechst 33342 (Invitrogen). Cell images were acquired with the laser scanning confocal microscope, ZEN Digital Imaging for LSM 700 (Zeiss), and analyzed using Zen 2010b SP1 imaging software (Zeiss) and ImageJ. For measuring PrP signals, an identical threshold was applied to all images. Then intensities of PrP signals and clustered particles were measured using ImageJ software.

Cells were fixed in 4% paraformaldehyde and 4% sucrose and permeabilized or not for 10 min with 0.2% Triton X-100. After being blocked for 30 min in PBS containing 3% bovine serum albumin and 0.3 M glycine, the cells were incubated overnight with anti-PrP SAF83 antibody (Cayman; 1:2000 dilution) or Sha31 (Spi-Bio) and anti-β-tubulin (Novus Biologicals; 1:2000 dilution), followed by incubation with fluorescently conjugated secondary antibodies (Invitrogen). For the resistance to denaturation, before incubation with antibodies, the cells were treated for 2 h at 4 °C with 3 M guanidine thiocyanate. Cells were scanned with a NanoZoomer 2.0RS scanner and analyzed using NanoZoomer digital pathology software (Hamamatsu Photonics). For confocal analyses, cells were plated on poly-d-lysine (Sigma)-coated microscope cover glasses (Thermo Fisher Scientific). Cell images were acquired with the laser scanning confocal microscope, ZEN Digital Imaging for LSM 700 (Zeiss), and analyzed using Zen 2010b SP1 imaging software (Zeiss) and ImageJ (https://imagej.nih.gov/ij/). For measuring PrP signals, an identical threshold was applied to all images. Then intensities of PrP signals and clustered particles were measured using ImageJ software.