Antibody Elution Protocol for Multiple Immunohistochemistry Staining

NOTE: Elute antibodies from fluorescently labeled slides to perform bright-field staining. One
can also elute antibodies from lightly stained bright-field slides to perform fluorescent staining.
Always have extra slides for positive controls and to make sure primary antibodies have been
removed.
1. Obtain slides whose primary antibody(ies) need to be eluted
2. Place slides in a glass rack (fits 19)
3. Place glass rack (s) in TBS and leave on shaker overnight to remove coverslips
4. The coverslips should have come off the slides overnight. If not, try to remove them
without tearing the tissues as they will be easy to remove
5. Once coverslips are removed, place slides in a glass rack again and follow as listed
below (all with shaking):
a. 10 minutes in TBS
b. 10 minutes in TBS
c. 10 minutes in TBS; tilt rack sideways to remove as much TBS as possible prior to
the next step. These steps should remove most of the glue used to coverslip
d. 1 hour in glycine SDS pH 2
e. 30 minutes in 0.1 M PBS (10X PBS)
f.

10 minutes in 1X PBS

g. 10 minutes in 1X PBS
h. 10 minutes in 1X PBS
6. If unsure that antibodies have been eluted, place extra slides in the secondary antibody
(ies) used to stain them previously and continue with either bright-field or fluorescent
protocol
7. If elution was successful, continue with staining protocol
Solutions:
Glycine SDS pH 2
-

Mix 0.94 g glycine in 25 mL 20% SDS, and fill to 500 mL with ddH 2O (enough for two
glass racks)

Adjust pH to 2 by adding concentrated HCl