Sep 21, 2021

Public workspaceStaining and imaging of mouse submandibular ganglion by α-bungarotoxin and nanosensor

DOI
dx.doi.org/10.17504/protocols.io.wxrffm6
  • 1Northeastern University
  • SPARC
    Tech. support email: info@neuinfo.org
Junfei Xia
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DOI: dx.doi.org/10.17504/protocols.io.wxrffm6
Protocol Citation: Hongrong Yang, Junfei Xia, James Monaghan, Heather A Clark 2021. Staining and imaging of mouse submandibular ganglion by α-bungarotoxin and nanosensor. protocols.io https://dx.doi.org/10.17504/protocols.io.wxrffm6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: January 09, 2019
Last Modified: September 21, 2021
Protocol Integer ID: 19153
Abstract
This protocol describes how to label acetylcholine receptors in isolated submandibular ganglion from mice using α-bungarotoxin and nanosensor formulated by the Clark lab.
Materials
MATERIALS
Reagent100% formalin Merck MilliporeSigma (Sigma-Aldrich)Catalog #F1635-500ML
Reagentα-Bungarotoxin Alexa Fluor 647 conjugatedThermo Fisher ScientificCatalog #B35450
ReagentPBS pH 7.4Thermo Fisher ScientificCatalog #10010023
ChATBAC-eGFP transgenic mice from The Jackson Laboratory.
Stock number: 007902
Protocol materials
Reagent100% formalin Merck MilliporeSigma (Sigma-Aldrich)Catalog #F1635-500ML
Reagentα-Bungarotoxin Alexa Fluor 647 conjugatedThermo Fisher ScientificCatalog #B35450
ReagentPBS pH 7.4Thermo Fisher ScientificCatalog #10010023
Reagentα-Bungarotoxin Alexa Fluor 647 conjugatedThermo Fisher ScientificCatalog #B35450
Extraction of submandibular ganglion
Extraction of submandibular ganglion
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.

Expected result
Well isolated salivary ducts with at least one submandibular ganglion attached


Staining of submandibular ganglion by α-bungarotoxin and nanosensor (formulated by Clark Lab)
Staining of submandibular ganglion by α-bungarotoxin and nanosensor (formulated by Clark Lab)
1. Rinse submandibular ganglion with PBS for Duration00:20:00
2. Soak submandibular ganglion in Amount500 µL Concentration0.000002 Molarity (M)
Reagentα-Bungarotoxin Alexa Fluor 647 conjugatedThermo Fisher ScientificCatalog #B35450
for Duration00:30:00 .
Alternatively, soak submandibular ganglion in Amount500 µL nanosensor solution formulated by Clark Lab for Duration00:30:00
3. Rinse submandibular ganglion with PBS for Duration00:20:00 .



Imaging of submandibular ganglion
Imaging of submandibular ganglion
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
ZEN pro
NAME
ZEISS
DEVELOPER