Re: fluorescein

From:"C.M. van der Loos"

Would it not be better to trace something that is non-fluorescent but 
certainly survives the fixation procedure? I mean something like 
digoxigenin that is also easy coupled to any protein (like FITC-
coupling. Via an anti-DIG/FITC antibody this hapten can be traced 
again. I believe there is a considerable risk that any fluorochrome can 
damaged by fixation techniques.
Chris van der Loos
Dept. of Cardiovascular Pathology
Academical Medical Center
Amsterdam - The Netherlands 

>Date: 21 Jul 2003 11:50:42 -0500
>From: Gayle Callis 
>Subject: Re: fluorescein
>Storage should be in the dark, to prevent photo bleaching of FITC when
>samples are collected, fixed, handled - etc.    After cutting sections 
>must be in dark after mounting with antifade mounting media.  Vector 
>has a nice one, Vectashield Hardset.  
>She can probably do all three preparations, but remember that if she 
>fixes in formalin or paraformaldehyde, the tissue will have some
>autofluorescence.  If the FITC signal is stronger, brighter than then
>dimmer autofluoresence, then all should go well.  In general, the
>microcapsules are huge! and very bright. 
>I would think ingesting FITC might damage it (digestive enzymes, etc) 
>but not sure, there may be some pH and enzyme considerations.  Have 
>her contact tech services at Molecular Probes, they usually have 
>answers for fluorochromes. 
>We have used FITC labelled microcapsules injected into bovine tonsil,
>removed tissue, snap freeze and cryosectioned and fixed sections
>immediately with NBF. Some autofluorescence occurs but avoid acetone, 
>it damages FITC or rather causes loss of fluorescence. After section 
>NBF fixation, rinse with PBS, sections were mounted with aqueous 
>mounting media compatible for fluorescent molecules. What little 
>autofluorescence occured was dim, plus looking at both red and green 
>channel gave interesting results, with autofluorescence appearing 
>reddish and microcapsules still bright yellow-green. It was a nice way 
>to use autofluorescence as contrast. So examine sections with both 
>excitations for FITC and Rhodamine, you get interesting results, and 
>be sure to examine with colocalization for those interesting results. 

>At 11:40 AM 7/17/2003 +0200, you wrote:
>>Dear List-Members
>>Not really an Histology question, but related. A colleague, who is 
>>working with diets for fish larvae wants to put fluorescein (FITC)as 
>>a marker in the food (microcapsules) for fish larvae, and after to 
>>measure the fluorescence. She would like to store the samples in some 
>>way until the measurements of fluorescence. The question is, does 
>>fluorescein would be afected by the storing of the sample? (she said 
>>to me she could freeze, freeze-dry, or maybe fix, in this last case, 
>>what would be the best fixative?). I have never worked with 
>>fluorescence, so I decided to ask the experts. Any advice will be 
>>thanks in advance 
>>Jos#233# Luis

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