Re: [Histonet] Autofluorescence blocking ---- LONG REPLY

From:"Gayle Callis"

One of the best reviews of autofluorescence and how to get rid of it in 
found on www.IHCworld website.  There is a link to a wonderful discussion on 
this from a group in Toronto if you go to to the fluorescence topic in 
IHCworld.  I also have a review of autofluorescence pertaining to GFP but it 
applies to immunofluorescent staining too.  If you want that, I will forward 

The Toronto group discusses the different sources of autofluorescence from 
naturally occuring to what is induced by aldehydes, plus suggestions for 
eliminating each type.  If you want, I can also forward this pdf to you.

I have put together a document from all Histonet responses pertaining to 
this problem, so forgive the long cut and pasted, sometimes repeated 
information below. We use the 100 mM glycine method, on rehydrated sections 
for 20 minutes (flood section as you would with an antibody).  I have seen 
300 mM glycine used also, at same pH and buffer.  100 mM has worked well for 

Good luck and hope you have the proper glowing results.  I have not given 
recognition to the gentleman who initially put this together, but I am 
grateful he did it.

Gayle M. Callis
Bozeman MT

1. After rehydrating your (formalin fixed or paraformaldehyde fixed paraffin 
embedded sections, OR NBF or PFA fixed frozen sections) and before blocking 
for protein incubate them in 100 mM glycine for 20 minutes.  This will 
quench autofluorescence caused by free aldehydes. Works like a charm, I use 
it .  2. I've pretty much moved away from doing IF on paraffin embedded 
stuff (ICC yes) but I have had some experience trying to knock back 
autofluorescence (endogenous and fix related) in tissue sections.I've had 
some luck pre-treating fixed sections w/ one of the following:1.  50mM 
Ammonium chloride in PBS for 10 min.2.  0.1M Glycine in PBS, pH 7.4 for 5-10 
min.3.  1% Sodium borohydride in PBS for 10-20 min. It varies from sample to 
sample which method works the best but I've had the most success w/ the 
borohydride and NH4Cl methods.Autofluorescence can be brought on by certain 
endogenous tissue constituents, ie. fibronectin, lipofuscin and elastin, as 
well as by fixation in aldehydes. You don't say if your sections are fixed 
or not. If so, you should look at using sodium borohydride (0.5mg/ml in PBS) 
for 5 minutes (glutaraldehyde) or PBS plus a few drops of 1M 
glycine(formaldehyde) to block any reactive  groups. ***Sodium borohydride 
is flammable on contact with water, and harmful by ingestion, inhalation 
etc.  Take adequate precautions*** Another thing to consider is reducing the 
section thickness, if possible,  as the intensity of autofluorescence is 
related to this.  You also don't mention what fluorochromes you are using. 
It may be   worthwhile trying a fluorochrome of a longer wavelength as there 
is less  likelihood of any spectral overlap with the endogenous material. As 
I   mentioned in an earlier posting today. we have had good results 
switching  to the Alexa dyes (Molecular Probes).  There are a couple of 
simple things you can do to help reduce autofluorescence.Some of the 
chemical reactions causing autofluorescence occur most rapidly with higher 
temperatures and on exposure to light. Therefore, performing the labelingat 
4 C in the dark can help reduce this problem.Autofluorescence intensity is 
related to section thickness. You may want to try thinner sections if at all 
possible. Sometimes using fluorophores excited at longer wavelengths can 
help diminish autofluorescence.If autofluorescence is still an issue, there 
are a few preincubation steps you could try. A Tris-glycine mixture (adjust 
0.1M glycine to pH 7.2-7.4 with 1M Tris base) will saturate free aldehyde 
groups. (15-30 minutes at room temp in Tris-glycine.Wash well in PBS.  The 
use of 1% sodium borohydride in PBS helps reduce any free aldehyde groups in 
the tissue, making them non-reactive. Incubate sections for 30 minutes 
inborohydride and then wash well(minimum 15 minutes) in several changes of 
PBS.Proceed with labeling. These techniques can be used alone or 
sequentially. If the tissue is fragile though, only use the Tris-glycine 
method.  Please note that sodium borohydride is very reactive and is 
flammable on contact with water.  Another technique to block unreacted 
groups is to incubate sections for 5 minutes in 50mM NH4Cl, and rinse in PBS 
before labeling.
----- Original Message ----- 
From: "Troutman, Kenneth A" 
To: "Histonet" 
Sent: Tuesday, July 15, 2008 4:12 PM
Subject: [Histonet] Autofluorescence blocking

Dear Histonetters,

I read awhile back about a reagent that you can use to block 
autofluorescence in tissue, but I cannot find the histonet string that 
contains that info.  Could someone remind me what that is and how you use 

Thank you!

Ashley Troutman BS, HT(ASCP)QIHC
Histopathology Laboratory
Department of Pathology
Vanderbilt University Medical Center
Nashville, TN

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