Staining and imaging of mouse submandibular ganglion by α-bungarotoxin and nanosensor

1. Enthanize animals using CO2.

2. Make a midline incision in the neck and expose the salivary glands.

3. Separate the pair of salivary glands and expose the salivary ducts.

4. Remove connective tissues surrounding salivary ducts and cut the ducts from where they enter salivary glands to where they enter digastric muscles. The submandibular ganglion are attached to cutted ducts.

1. Rinse submandibular ganglion with PBS for 0h 20m 0s

2. Soak submandibular ganglion in 500µL``0.000002Molarity (M)

for 0h 30m 0s.

Alternatively, soak submandibular ganglion in 500µLnanosensor solution formulated by Clark Lab for 0h 30m 0s

3. Rinse submandibular ganglion with PBS for 0h 20m 0s.

1. Place the submandibular ganglion on a glass slide and cover it with cover slip.

2. Image the whole tissue using a Zeiss LSM 700 confocal. Use 488 nm laser line for GFP and Oregan Green 488 channel, 555 nm laser line for pHAb, and 639 nm laser line for bungarotoxin-Alexa Fluor 647 channel. Set laser power at 2% or at the appropriate power intensity that cellular structures can be clearly seen.

Software

Value	Label
ZEN pro	NAME
ZEISS	DEVELOPER
https://www.zeiss.com/microscopy/us/products/microscope-software/zen.html	LINK