May 19, 2026
Quenching autofluorescence with Cupric Sulfate
- 1University of Alabama at Birmingham
Protocol Citation: Laura Volpicelli-Daley, Marissa Menard 2026. Quenching autofluorescence with Cupric Sulfate. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjkk4ngk5/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 15, 2026
Last Modified: May 19, 2026
Protocol Integer ID: 317287
Keywords: ASAPCRN, lipofuscin autofluorescence from brain section, quenching autofluorescence with cupric sulfate, lipofuscin autofluorescence, other methods for quenching autofluorescence, quenching autofluorescence, fluorescence from secondary antibody, desired fluorescence, cupric sulfate, human brain tissue, secondary antibody
Funders Acknowledgements:
ASAP
Grant ID: 020616
Abstract
This protocol describes how to quench lipofuscin autofluorescence from brain sections. The protocol works for mouse and human brain tissue. We find this method, unlike other methods for quenching autofluorescence, does not diminish desired fluorescence from secondary antibodies.
Materials
Reagents required to prepare Cupric sulfate solution:
- 10 mM CuSO4
- 50 mM ammonium acetate, pH, 5.0
- Store indefinitely at 4°C
Protocol materials
TBS
mounting media
Cupric sulfate solution
Protocol
1h 25m
Perform after secondary antibody incubation and 3 times 00:05:00 rinses with TBS .
(00:05:00 , 00:05:00 )
15m
Place Cupric sulfate solution into wells of a six well tissue culture plate. Transfer the tissue sections into the wells and incubate at 4 °C with shaking for 00:30:00 .
Note
This step reduces autofluorescence from lipofuscin granules in the tissue (Schnell et al., 1999).
30m
Transfer the tissue to the wells of a tissue culture plate containing TBS . Rinse the tissue three to four times in TBS with shaking for about 00:10:00 .
( 00:10:00 , 00:10:00 , 00:10:00 ).
40m
Mount tissue on slides and coverslip with mounting media .
Protocol references
Schnell, S.A., Staines, W.A., and Wessendorf, M.W. 1999. Reduction of lipofuscin-like autofluorescence in fluorescently labeled tissue. J. Histochem. Cytochem. 47:719-730.