A1 confocal laser scanning microscope system

For immunofluorescence assay, brains tissues from each group were harvested and then immersed in 4% formaldehyde solution for 24 h followed by gradient of dehydration in 10, 20, and 30% of sugar solution. The brain tissues were OCT-embedded and sliced into coronal sections by a frozen microtome (Leica CM1900, Wetzlar, Hesse-Darmstadt, Germany) at a thickness of 10 μm through the dorsal hippocampus. After the sections were permeabilized with PBS/0.1% Triton × 100, the sections were incubated in the mixture anti-NeuN (mouse, Abcam, 1:1000) and anti-BDNF (rabbit, Abcam, 1:750) at 4°C overnight, and then followed by fluorescence secondary antibody, including DyLight 488 affiniPure goat anti-mouse IgG (green) and DyLight 594 affiniPure goat anti-rabbit IgG (red). Then, the sections were washed with PBS and mounted by DAPI. Finally, the pictures were captured by a Nikon A1 confocal laser scanning microscope system (Nikon Corp., Tokyo, Japan).

The Ag-implanted PAAm hydrogel is sectioned into ∼60 μm thick slices (Vibratome 1000) to expose the cross-sectional profile and subsequently loaded into a super critical point dryer (Samdri 780A) to remove the water content without causing significant structural change to the hydrogel. The dried slice is then glued on a metal plate and examined directly with a Hitachi S-4700 FE-SEM (field emission scanning electron microscope) under a voltage of 10–20 kV. For confocal microscopy, a bulk piece of Ag-implanted PAAm gel is placed on top of a glass side, scanned with a × 60 water immersion lens and imaged using a Nikon A1 confocal laser scanning microscope system on a Nikon Eclipse 90i using the 561.4 nm excitation laser line (Nikon Instruments Inc., Melville, NY).

The mutated PT2 was cloned into the binary vector 1300UR at the BamHI and KpnI sites and was introduced into N. benthamiana via agrobacterium-strain-GV3101-mediated transformation. Fluorescence signals were monitored using a Nikon A1+ confocal laser scanning microscope system (Tokyo, Japan). At least three independent biological repeats were conducted with similar results.