[Histonet] glycine method to remove aldehyde induced autofluorescence

[Gayle Callis]

Dear All, 

 

Some person kindly mentioned my name as a source for the glycine method to
remove aldehyde induced autofluorescence.  We liked the simplicity of this
method, plus gentle to tissue sections.  

 

This was the original information but we modified it.   I have seen
concentrations of glycine range from 100 mM to 700 mM . 

 

Original method:  
 
1. Rehydrated tissue sections:  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 for FFPE sections. Wash well in PBS.
If the tissue is fragile though, only use the Tris-glycine method.
 
2..  For tissues coming out of formalin, soak the tissues for 30 min to 1
hour and rinse well. 

 

 

Callis Modified Method: 

 

We did not use TRIS-glycine, preferring the same buffer used in IF staining.
Make fresh for a day's use.   500 mM glycine in pH 7.2 - 7.4  Dulbeccos PBS
(Sigma).  We increased concentration to 500 mM glycine for 15 - 30 minutes
at RT after we found 100 mM reduced autofluorescence while the higher
concentration did a better more complete removal.    I don't think it makes
much difference if you use TBS or DPBS so you can use whatever your lab
prefers for IHC/IF staining should work equally well.     

  

We found  two changes of glycine solution worked well since you are
refreshing the solution to sop up those free aldhydes.  Do 15 minutes
incubation for each change, don't  rinse the sections between changes, just
tip, drain slides, blotted edges of sections, add solution on sections with
slides laying flat on a manual stainer.   Some people might prefer glycine
solution in a coplin jar if they are going to an automated staining system.


 

If you fear drying, one method was 700mM glycine, 0.15% BSA (use pure IgG
and protease free), and 0.1% sodium azide in PBS with 15 to 30 min RT
incubation.   Sodium azide can be left out since it is there to prevent
bacterial growth, and deemed unnecessary since our glycine solution was made
fresh before a one day/one time use.  Glycine is cheap and goes into
solution readily.  

 

FYI, lysine has also been used to get rid of free aldehydes (Elias J.
Immunohistopathology book) 

 

Good luck

 

Gayle Callis

HTL/HT/MT(ASCP)  

Bozeman MT