Metamorph 5

Brains were removed from adult mice and post-fixed, overnight. After de-hydration, brain specimens were embedded in Paraplast Tissue Embedding Medium (Leica Biosystems, Milan, Italy) and serially sectioned to obtain sagittal sections (thickness, 9 micron) [46 (link)] that were then immunostained as previously described [38 (link)]. Briefly, sections were incubated with the anti- Aβ monoclonal antibody DE2B4 (Acris, Herford, Germany), which recognizes Aβ aggregates of senile plaques), at 4 °C overnight. After several washes with PBS, sections were incubated with biotinylated secondary antibody and antibody-antigen complexes were visualized using the Vectastain ABC Elite and DAB Peroxidase Substrate Kits (Vector laboratories, Burlingame, CA, USA), according to manufacturer’s instructions. Adjacent microscopic fields encompassing the cortical and hippocampal regions were acquired using the MetaMorph 5.5 software (Universal Imaging, Downgtown, PA, USA). The abundance of Aβ plaques per field was determined blindly and independently by two investigators and relative plaque area was measured using the MetaMorph 5.5 software tools (Universal Imaging, Downingtown, PA, USA).

Larvae within 2 days dph were used for exposure. Twenty larvae were exposed in a 50 mm glass Petri dish for 24 h. Triplicates were carried out for each concentration and for the solvent control. After exposure, the fish were deposited onto the inner side of Petri dish cover with little water left. The induced GFP expression in the liver was observed under stereomicroscope (model M205 FA, Leica Microsystems, Germany). The images were recorded with the same parameter settings for the same set of exposed samples. Fluorescence intensity was quantified by image analysis software, Metamorph 5.0 (Universal Imaging, Downingtown, PA, USA). To reduce the interference of fish auto-fluorescence, only the fish liver was selected for analysis. The average fluorescence of the liver area was regarded as the GFP signal intensity. All procedures with fish were conducted following the License to Conduct Experiments approved by the Government of the Hong Kong SAR Department of Health [Ref. (21) in DH/HA&P/8/1/1 Pt.3].

Another set of Z. mays seedlings was treated with 100 nM LatB, 500 nM eBL and the solvent control solution and mounted on square Petri dishes following the methods described above. The plates were positioned so that the roots were in a vertical orientation. After 60 min, the plates were rotated 90° so that the roots were horizontal. Immediately after rotation, images were captured every 15 min for 12 h. Time-lapse movies were generated using the Metamorph 5.0 image acquisition software (Universal Imaging Corp, USA). From the generated time-lapse movies, the root tip angle with respect to the horizontal was measured using ImageJ software. Experiments were repeated at least three independent times with 15-20 seedlings per treatment.