Re: Fluorescent immunohistochemistry

<< Previous Message | Next Message >> (Karen Larison),

Hello Histonetters, Hello Jennifer Johnson,

I used to do fluorescence IHC product development at Molecular Probes, so will 
attempt to answer most of your questions:

First, Molecular Probes includes a good reference list in their catalog on 
fluorescence techniques.  It's probably also on their Web site:

Second, here's a question for you:  are you attempting to do double-labeling with 
primaries of the same or different species?  If the primaries are from different 
species, life is a piece of cake.  You can incubate both primaries simultaneously, 
then wash, then inubate both secondaries simultaneously.  If you are attempting to 
double-label two primaries from the same species, it's much more difficult, and the 
answer is much more involved.  So let me know if you need further advice on this 

Other answers to other questions follow:

1.  I have worked out the concentration of antibody needed for both paraffin
and frozen tissues with the ABC systems.  If the antibody works for alk phos
and peroxidase, will it also work for fluorescence?

I haven't done any work with paraffin sections, but it's my understanding that 
paraffin will exacerbate autofluorescence problems.

On frozens, you use the same concentration of primary antibody as you would with the 
chromogenic system.

2.  Will the dilutions for the antibody be about the same or is there some
difference in the inherent in either the precipitating chromagen system and
the fluorescent system that makes one system work at a lower dilution?

Each fluorophore is several times more detectable than any chromophore.  However, any 
good commercial fluorescent antibody will contain only 2-3 fluorophores. If you 
conjugate more fluorophores to the antibody, the fluorophores will self-quench and 
the antibody will tend to denature, thereby increasing the background.  In an 
enzyme-mediated system such as ABC, you get a much greater quantity of chromophore 
deposited per antibody than the 2-3 fluorophores/antibody in a fluorescence system.  
In theory, the enzyme-mediated system should be more sensitive.  In practice, with 
most antibodies, I don't believe this is true.  If you have a good fluorophore on 
your secondary antibody, in most cases, the two systems will be approximately 
equivalent in sensitivity.  It will depend on the primary antibody.  Probably this is 
because fluorophores behave differently in different microenvironments.  For 
instance, the Bodipy fluorophore bleaches quite badly in some aqueous environments, 
whereas it is doesn't bleach much at all in lipophilic environment.

  2.  I am thinking about using a secondary that is already fluorescently
tagged, but without the amplification of the biotin system, is this type of
fluorescence system less sensitive?

The best of both worlds may be the fluorescent tyramides, which are fluorescent HRP 
substrates.  They are truly more sensitive than either the chromogenic systems or the 
fluorescently tagged secondaries, but they have other issues.  If you need enhanced 
sensitivity, they are certainly worth trying.  However, in most circustances, the 
fluorescently tagged secondaries do just fine, so why go to the extra work and 
expense?  Also, you don't get a noticeable increase in sensitivity by going to a 
biotinylated secondary/fluorescently tagged avidin system.

3.  Are the needs for blocking different?  
	Using the systems from Vector, depending on the antibody, I had to
perform some/all blocking listed below which differed per kit:  
		Alk Phos - Levamisole, Normal goat/horse serum and
		Peroxidase - peroxidase quench, Normal goat/horse serum and
		Sometimes in either system I had to use an Fc receptor block

Usually, the serum and Fc receptor blocks will suffice, except with the tyramides, 
which may come with its own proprietary blocking agent.

4.  How long do you "develop" or incubate the fluorescent tag?  Does leaving
it on longer give background like leaving DAB on too long?  

A fluorescently tagged secondary will require an approximate half-hour incubation at 
room temperature, followed by copious washes.  Longer incubations will not ordinarily 
increase the background, assuming the antibody is good and the dilution is correct.  
The correct dilution for most fluorescently tagged secondary antibodies is 
approximately 10 micrograms/mL.

The fluorescent tyramides behave more like DAB; 3-10 minutes incubation will suffice.

5.  Are there any restrictions on which buffers or solutions that may quench
the fluorescence or otherwise ruin the experiment?  Are there any
recommended buffers or solutions that I have to use to obtain staining?

Fluorescein is much brighter at a slightly alkaline pH.  I recommend using a Tris 
buffer, pH 8 with fluorescein-tagged secondaries.  Most fluorophores are not 
sensitive to pH changes.  Fluorescein is the exception.  Most fluorescently tagged 
secondaries can be used in either phosphate or tris buffers, whichever is convenient.

6.  Any suggestions on which tags work best?  I have been reading about
Alexa dyes and was thinking of using two that were far enough apart, Alexa
488 and 594.  Any experience with Alexa dyes?

What filter sets do you have?  I have used the green Alexa 488, used with a standard 
fluorescein filter set, and the red Alexa 546, used with the standard rhodamine 
filter set.  My eye couldn't detect a difference in a side-by-side comparison of 
fluorescein- vs Alexa 488-tagged secondaries.  However, in some circumstance, 
fluorescein-tagged secondaries may have severe photobleaching problems, particularly 
if you are doing confocal microscopy.  In general, fluorescein is the brightest 
fluorophore available, but if you're planning to do confocal microscopy, I would use 
the Alexa 488.  In contrast, the Alexa 594 gives a much brighter signal than the 
rhodamine secondaries.  The Alexa 594 is fantastic.

In general, autofluorescence is less of a problem at longer wavelengths.  So if this 
is a problem, and you have the correct filter set, try a longer wavelength red Alexa 
7.  What are some of the major problems that I might encounter with
fluorescent labeling? 

It takes some getting used to, because you can't see the tissue.  Also, the signal is 
sometimes not very bright under lower power, so it takes some time to find the signal 
if you are unfamiliar with the location of the signal.  Counterstains such as 
propidium iodide or DAPI will help to get you around the tissue until you are in the 
right locale.  I prefer fluorescence (because it's quicker and easier and beautiful) 
with my old familiar antibody friends where I'm familiar with the staining pattern.  
If I'm unfamiliar with the staining pattern, I prefer HRP/DAB cause it's easier to 
locate the signal.

Again, if you are double-labeling with same-species primaries, fluorescence 
techniques can be problematic.  If that's the case, let me know, and I'll provide 
some guidance in this arena.  

8.  Are there methods to quench autofluorescence in tissues? 

I have found that in fish tissue, autofluorescence becomes quite problematic if the 
tissue is dehydrated, so I mount in a 50:50 glycerol/buffer system, and seem to have 
no problem.  Fluorophores, including endogenous fluorophores (like the flavins), are 
molecules with with extensive conjugated pi-bonding systems.    One method for 
reducing autofluorescence, is to pretreat the tissue with 0.1% sodium borohydride in 
PBS (Cytometry 8, 235, 1987), which effectively reduces the number of pi bonds in the 
tissue, thereby decreasing the autofluorescence.  The other trick, as mentioned 
above, is to use the longer wavelength fluorophores.

If you have further questions, please don't hesitate to ask.

Karen in Oregon

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