Ab150170

The expansion microscopy technique [109 ] optimized for Arabidopsis seeds was conducted as previously described [73 (link)]. Anti-Fibrillarin antibody (ab4566, Abcam) and anti-alpha Tubulin antibody (ab89984, Abcam) were used in 1:500 dilution as primary antibodies. Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) (ab150113, Abcam) and goat Anti-Chicken IgY H&L (Alexa Fluor® 555) (ab150170, Abcam) were used in 1:500 dilution as secondary antibodies. For each sample, a stack of nine images with 1-μm intervals were recorded by ZEISS LSM700 with 25× oil objective and ZEN software at 1024 × 1024 resolution in 8-bit. DAPI signals were excited by 405-nm laser and passed through SP490 filters. Alexa488 signals were excited by 488-nm laser and passed through BP490-635 filters. Alexa555 signals were excited by 555-nm laser and passed through 560–1000-nm filters. Pinhole sizes were kept as 1 airy unit for each color, and color channels were scanned separately. FIJI software was used for image processing and nuclear size quantification. Each stack of images was first Z-projected on maximum intensity and then the nuclear areas were determined based on DAPI signals. The zygotic nuclei were distinguished from the endosperm nuclei according to position and tubulin staining patterns.

Sixteen-μm-thick slices of brain tissue from Wistar and OXYS rats were put on Polysine-glass slides (Menzel-Glaser, Braunschweig, Germany) and kept in a blocking solution consisting of 1% BSA (Sigma–Aldrich, St. Louis, MO, USA) in PBS with 0.1% of Triton X-100 (PBS-T) for 1 h. Next, the slides were probed at 4 °C overnight with the primary antibody that was used for the western blot analysis and with an antibody against the amyloid β peptide (cat. # MABN254; MOAB-2; Merck Millipore, Darmstadt, Germany) diluted with the blocking solution at 1:250. After a few washings with PBS, the slides were treated at room temperature for 1 h in the dark with a secondary antibody (1:5000; cat. # ab175472, ab150170, and ab150075; Abcam, USA) diluted in a 1:300 ratio with PBS containing 1% of BSA. Then, the tissue sections were washed in PBS and coverslipped by means of a mounting medium containing 4′,6-diamidino-2-phenylindole (cat. # ab104139; Fluoro-shield with DAPI; Abcam, USA). The negative controls were subjected to the same processing except there was no primary antibody. The profiles of immunofluorescence signals were observed by fluorescence microscopy (Axioplan 2; Zeiss, Oberkochen, Germany).

This procedure was performed by a standard indirect method as described previously [8 (link)]. Primary antibodies and dilutions were as follows: anti-VEGF (1:250; # ab14078, Abcam) and anti-amyloid-β_1–42 (1:250; # ab10148, Abcam). After incubation with the respective secondary antibodies conjugated with Alexa Fluor 555 or Alexa Fluor 488 (## ab150170, ab150073, Abcam) diluted 1:250, the tissue slices were coverslipped with the Fluoro-shield mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI; # ab104139, Abcam) and examined under an Axioplan 2 microscope (Zeiss).