Reducing Autofluoresence in Cells and tissue.

Chemical Reduction to
Minimize or Eliminate Autofluorescence
These are a few protocols for reducing Autofluorescence in cells and tissue. 

Sodium Borohydride

This is perhaps the most
widely used method for autofluorescence reduction in tissue sections.

[Protocol derived from
Jennifer Kramer and similar protocol described in Beisker et al., 1987]

Below is a protocol for
reducing autofluorescence. It is applicable for both tissue sections and for
monolayers of cells. This protocol is intended to be applied after tissue
rehydration and permeabilization but directly before any blocking steps or
incubations with other probes (antibodies, dyes, etc.).

1. Immediately before
use, make up a 1 mg/mL solution of sodium borohydride in a physiological buffer
such as PBS. The solution will be fizzy like carbonated water.

2. Apply this solution
immediately (while fizzing) to cells or tissue sections.

 

Incubation Times.

For tissue culture cell
monolayers fixed in glutaraldehyde, incubate in the sodium borohydride solution
for 4

minutes. Replace with
fresh sodium borohydride solution and incubate for another 4 minutes.  For
paraformaldehyde fixed, paraffin embedded tissues at 7 um thick or less,
incubate 3 times, 10 minutes each in sodium borohydride solution. For thicker
tissue sections or whole mount flies/worms/ baby fish/ etc., more changes of
sodium borohydride solution and/ or longer periods of incubation might be
necessary.

3. Rinse many times with
buffer or physiological saline to remove traces of sodium borohydride.

4. Continue with
blocking steps at this point. Discard any leftover sodium borohydride solution
as it loses its reactivity with time.

Sodium cyanoborohydride
is alternative, but generates cyanide as a by-product. [submitted by Dr.
Mario M. Moronne (NanoMed Technologies) from the Confocal Archives. ]

 

References:

Beisker W. Dolbeare F.
Gray JW. An improved immunocytochemical procedure for high-sensitivity
detection of incorporated bromodeoxyuridine. Cytometry. 8(2):235-9, 1987.

Clancy B. Cauller LJ.
Reduction of background autofluorescence in brain sections following immersion
in sodium borohydride. Journal of Neuroscience Methods. 83(2):97-102, 1998.

Mosiman VL, Patterson
BK, Canterero L, Goolsby CL. Reducing cellular autofluorescence in flow
cytometry: an in situ method. Communications in Clinical Cytometry,
30(3):151-156 (1997).

Tagliaferro P. Tandler
CJ. Ramos AJ. Pecci Saavedra J. Brusco A. Immunofluorescence and glutaraldehyde
fixation. A new procedure based on the Schiff-quenching method. Journal of
Neuroscience Methods. 77(2):191-7, 1997.

 

Methods of Quenching Autofluorescence

Commercial Reagents

Millipore sells
“Autofluorescence Eliminator Reagent” (Cat# 2160) which is intended to reduce
lipofuschincaused

neural tissue samples. Link: http://www.millipore.com/catalogue/item/2160.

 

Cupric Sulfate (1 to 10 mM CuSO4) in 50mM Ammonium
Acetate Buffer (pH 5.0)

Cupric sulfate has been
shown useful against lipofuscin autofluorescence in neural tissue; treat
10-90 min., dip in water, then wash in PBS. [S. A. Schnell et al. 1999.
Reduction of lipofuscin-like autofluorescence in fluorescently labeled tissue.
J. Histochem Cytochem. 47: 719-730.]

 

Evans Blue

A 50 ug/mL solution of
the dye, Evans Blue, for 10 minutes, has been used to combat green
autofluorescence contributed by catecholamine in paraffin sections, works by
absorbance quenching. [De la Lande IS, Waterson JG. 1968. Modification of
autofluorescence in the formaldehyde-treated rabbit ear artery by Evans blue. J
Histochem Cytochem. Apr;16(4):281-2]

 

Pontamine Sky Blue

The dye Pontamine Sky
Blue has been shown to be superior to Evans Blue in combating catecholamine
autofluorescence in paraffin sections with minimal interference with FITC
labels. Use 0.05% Pontamine sky blue in PBS containing 1% DMSO. [T. Cowen, A.J.
Haven, G. Burnstock. 1985. Pontamine sky blue: A counterstain for background
autofluorescence in fluorescence and immunofluorescence histochemistry.
Histochemistry 82: 205-208]

 

Sudan Black B

Works by spectral
quenching of autofluorescence, but can also quench specific dyes to some
extent. Good

for lipofuscin.
Not compatible with xylene-based mounting media. Not compatible with Fast Blue
or True

Blue staining of
neurons, due to elimination of signal.  [S. A. Schnell et al. 1999.
Reduction of lipofuscin-like autofluorescence in fluorescently labeled tissue.
J. Histochem Cytochem. 47: 719-730].

 

Trypan Blue

Trypan Blue has been
shown to be useful for increasing signal-to-noise ratio for FITC-labeled cells
in flow cytometry. Authors suggest usage for FITC or PE labeled cells, but not
for farther-red emitting dyes.  Optimum condition was determined to use at
1.0 ug/mL, after antibody labeling with probe of interest, in ice-cold Trypan
Blue solution for 10 minutes at 4oC, followed by brief
wash before imaging. [V.L. Mosiman, et al. 1997. Reducing cellular
autofluorescence in flow cytometry : an in situ method. Cytometry 30:
151-156.]

Photobleach Reduction of
Autofluorescence

Autofluorescence of
paraffin sectioned tissue can be reduced through photobleaching with mercury arc
lamp or UV lamp, for up to 48 hours (M. Neumann & D. Gabel. Simple method
for reduction of autofluorescence in fluorescence microscopy. J. Histochem
& Cytochem 50(3): 437-439), though the authors did not examine recovery of
autofluorescence. Reduction of autofluorescence in paraffin sections via
photobleaching outperformed Sudan Black B, CuSO4 in ammonium acetate
buffer, sodium borohydride, and Pontamine Sky Blue.

Sunlight can also be
used for this purpose, as demonstrated using arterial tissue, as well as UV
illumination: (K. Kingsley et al. 2001. Photobleaching of arterial
autofluorescence for immunofluorescence applications.  BioTechniques 30:
794-797). Solar radiation was at least 20% more efficient than UV radiation.
Regarding recovery of autofluorescence: after 10 days, 84-100% of
autofluorescence returned with UV-radiated samples, but solar samples had
recovered no more than 27%.

 

Digital Removal of
Autofluorescence

Post-Acquisition
Processing of Images (= “Image Arithmetic Method”)

Autofluorescence is
usually very broad in fluorescence. By leaving one channel open and unused by
any fluorescent dye (usually FITC or Texas Red channel), you can use the
imaging software to digitally subtract the image taken of the open channel from
the image taken of a channel with the dye label, leaving only the specific
signal. In cases where a channel cannot be left open, the process can still be
performed as long as there is no overlap between specific signals in the two
channels. This post-acquisition processing can be tricky, however, since you
have to accurately judge how much intensity to subtract. Otherwise the
resultant image can look too contrasted. Such processing should be made
apparent in resultant publication of images.  An example where this has been
used: Anda Cornea. 2001. Digital subtraction of lipofuscin’s fluorescence.
Microscopy Today. Issue 01-4: 14.

 

Spectral Deconvolution
Systems

Certain special
microscopy imaging systems allow spectral analysis by analyzing emission
spectra of dyes and comparing against a library of spectra. The dyes are then
separated by spectrum and re-colorized according to user preference for a
resultant image. A no-dye control of a sample will yield the autofluorescence
spectrum which can then be subtracted from the resultant image.  These
systems include the Zeiss 510 META and Leica SP2 confocal microscopes, the
Nuance widefield microscopy imaging system, and the Maestro in-vivo imaging
system.

Stephen Full, M.S.

Technical Application Scientist,
Supervisor

Molecular Probes Labeling &
Detection Technologies

T
(541) 335 0353  F (541) 335 0238

29851 Willow Creek Rd, Eugene, OR,
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