May 14, 2024
Enzymatic treatment of free floating sections
- 1Rush University
Protocol Citation: Bryan Killinger 2024. Enzymatic treatment of free floating sections. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbzyyqgpk/v1
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: May 14, 2024
Last Modified: May 14, 2024
Protocol Integer ID: 99781
Funders Acknowledgement:
Michael J. Fox Foundation
Grant ID: ASAP-021030
NINDS
Grant ID: 1R01NS128467
Abstract
General protocol for enzymatic treatment of free floating brain sections.
Prepare 40 micron thick free floating brain sections for enzymatic treatments.
Prepare 40 micron thick free floating brain sections for enzymatic treatments.
30m
Wash in DM
10m
Wash in DM
10m
Wash in DM
10m
Equilibrate tissue sections in reaction buffer.
Equilibrate tissue sections in reaction buffer.
10m
For GluC treatment, incubate in 1X GluC reaction buffer (New England Biolabs)
5m
For Trypsin treatment, incubate in TBS (50mM Tris-HCl, pH 7.6, 150mM NaCl)
5m
Enzymatic Treatment
Enzymatic Treatment
1d 8h
For Gluc, combine 100ul 2X GluC reaction buffer and 100ul GluC (New England BioLabs, 100 ng/µl dissolved in high-purity water). Incubate tissues with mixture at 37C for 16h.
16h
For Trypsin, combine 4ul of trypsin stock (Sigma-Aldrich, 1 mg/ml dissolved in 1 mM HCl, pH 3) and 196ul of TBS. Incubate tissues with the mixture at 37C for 16h.
16h
Wash
Wash
30m
wash tissue in DM
10m
wash tissue in DM
10m
wash tissue in DM
10m