[Histonet] Re: Histonet Digest, Vol 73, Issue 33

[James Watson]

We routinely quench RBC auto-fluorescence with the following procedure:

10 mM Copper Sulfate

10 mM Copper Sulfate
Cupric Sulfate...........................................1.25 gm
50 mM Amonium acetate (pH5)....................500.0 ml
Adjust ph to 5.0 with 1.0 M NaOH

50 mM Ammonium acetate (pH5)
Ammonium acetate.....................................1.93 gm
Distilled water............................................500.0 ml
Adjust pH to 5.0 with 1.0 M HCl

1.	After staining wash slides in Reaction Buffer 5 times for 10 minutes each.
2.	Rinse in PBS 2 times for 10 minutes each.
3.	Rinse in distilled water 5 minutes.
4.	Place slides in 10mM copper sulfate for 8 minutes.
5.	Return slides to distilled water and check for autofluorescence with microscope.
6.	if needed return slides to 10mM copper sulfate for a couple of more minutes and check again.
7.	Rinse slides for 5 minutes in distilled water.
8.	Rinse in PBS 2 times for 10 minutes each.
9.	Coverslip slides with appropriate mounting media.


-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of mesruh turkekul
Sent: Thursday, December 24, 2009 10:20 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Re: Histonet Digest, Vol 73, Issue 33

Hi Naira,



The RBCs have a heme groups that have very high autofluorescence. They will
glow with every filter in every color. They will glow even without any
staining.
Just try to image unstained section and you will see.

"*Autofluorescence* is the
fluorescence<http://en.wikipedia.org/wiki/Fluorescence>of other
substances than the
fluorophore <http://en.wikipedia.org/wiki/Fluorophore> of interest. It
increases the background signal.Autofluorescence can be problematic in
fluorescence
microscopy <http://en.wikipedia.org/wiki/Fluorescence_microscopy>. In most
fluorescence microscopy, fluorescent stains (such as fluorescently-labeled
antibodies <http://en.wikipedia.org/wiki/Antibody>) are applied to samples
to stain specific structures. Autofluorescence interferes with detection of
the resulting specific fluorescent signals, especially when the signals of
interest are very dim - it causes structures other than those of interest to
become visible. Depending upon the shape of the structures of interest and
the other structures, it may not be obvious that this has occurred. In some
microscopes (mainly confocal
microscopes<http://en.wikipedia.org/wiki/Confocal_microscope>),
it is possible to make use of different lifetime of the excited states of
the added fluorescent markers and the endogenous molecules to exclude most
of the autofluorescence."


Unfortunately there is no way to avoid that. If you check online you may
find people using:
sodium borhydrate, 0.1M Glycine pH=3, sudan black or many other reagents to
prevent autofluoresence but the success rate is very low.
You may try to get rid of the red signal of the RBC with the imaging
software. They have different spectra than Alexa594 and you can do spectral
separation.

Happy holidays!

Mesru
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