Enzymatic treatment of free floating sections
Bryan Killinger
Published: 2024-05-15 DOI: 10.17504/protocols.io.81wgbzyyqgpk/v1
Abstract
General protocol for enzymatic treatment of free floating brain sections.
Steps
Prepare 40 micron thick free floating brain sections for enzymatic treatments.
1.
Wash in DM
2.
Wash in DM
3.
Wash in DM
Equilibrate tissue sections in reaction buffer.
4.
For GluC treatment, incubate in 1X GluC reaction buffer (New England Biolabs)
5.
For Trypsin treatment, incubate in TBS (50mM Tris-HCl, pH 7.6, 150mM NaCl)
Enzymatic Treatment
6.
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.
7.
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.
Wash
8.
wash tissue in DM
9.
wash tissue in DM
10.
wash tissue in DM
