[Histonet] Fluorescent immunostaining versus IHC

Gayle Callis gcallis <@t> montana.edu
Tue Apr 12 14:52:24 CDT 2005


Our immunofluoroescent staining is commonly done with two fluorophores, and 
have done it for both confocal and fluorescence microscopy.  It isn't 
anymore difficult but there are some tricks to the trade, so to speak. We 
basically do it the same as IHC, but have noticed, at least with our 
antibodies, the primary antibody concentration increases.

We cannot use a fluorophore directly conjugated to FITC for our mouse 
frozen sections and apply these antibodies for fluorescence microscopy - 
these are murine CD4 FITC or CD8-FITC - we get NO fluorescence. There are 
reasons for this.  With human kidney biopsies and IFA staining, the 
staining is excellent with directly conjugated primaries - IgG-FITC, etc. 
although the frozen sections are fixed minimally with cold acetone.

  However, one can increase the fluorescence signal a great deal ( and you 
want the brightest signal with the least photobleaching you can get) by 
using biotinylated primaries then Strepavidin-Alexa dyes OR a purified 
antibody followed by a fluorophore conjugated secondary i.e. secondary 
conjugated to FITC or RRX or a rhodamine that is crisp, separates nicely 
from FITC, and is more resistant to photobleaching.

With our murine CD marker work, we prefer using a primary that is purified 
w/secondary antibody-fluorophore or a biotinylated antibody 
w/Strepavidin-Alexa dye (488 equivalent to FITC) or Strepavidin-555 
(equivalent to Rhodamine).   How we combine them for double IFA staining 

With any fluorophore conjugated secondary, it is a good idea to spin the 
diluted antibody just before applying to section to eliminate protein 
aggregates that have fluorophore attached  - we refer to this as glowing 

One can purchase secondary antibodies from Molecular Probes that are 
conjugated to Alexa dyes and we only use secondary antibodies adsorbed to 
the species being stained.

You need to be aware of autofluorescence and what causes it -  major 
culprits are NBF or paraformaldehyde fixation  - very annoying and Histonet 
abounds with questions on how to get rid of it, not much fun nor always an 
easy task.

We avoid aldehyde fixatives for this reason and use fresh frozen sections 
fixed with acetone or acetone/ethyl alcohol mixture.  We avoid methanol 
with CD markers. Some use paraformaldehyde or other fixtives but that must 
be determined with fixation panels.

I have never used a detection kit for IFA work, everything is inhouse 
dilutions of secondary antibodies or Strepavidin Alexas.  We do not use 
Strepavidin conjugated to any FITC or its derivatives, the rhodamines.  We 
found they did not hold up as well as Alexa conjugates.  FITC and FITC 
derivatives are fluoresceinated versus the Alexa dyes which are sulphonated 
- with the latter being more stable with less photobleaching when excited.

Our technics tend to be rather purist, and we generally leave Tween out of 
buffers,  although some do use it.  All incubations with the fluorophore 
conjugates are done in the dark, so if you use an open immunostainer, it 
must be covered or do simple manual staining for that portion. We now 
coverslip using Molecular Probes Prolong Gold antifade, hard set and ready 
to use.

Jackson ImmunoResearch has excellent antibodies - get their hard copy 
catalog - a wealth of information. Rockland and Southern Biotechnology are 
two more antibody sources. Molecular Probes has a wonderful handbook 
although huge, but it is very educational and FREE.   Many companies 
recommend freezing down aliquots of fluorophore conjugates, and their spec 
sheets tell how to do this.

This is a good book both IHC and IFA information:

Immunochemistry 2, a practical approach Edited by AP Johnstone and MW 
Turner, ISBN# 0 19 963609 5, paperback.  Purchased from Springer-Verlag 
publishers, they have a website.

very you wrote:
Hi Renee,

I do confocal microscopy rather than on paraffin sections but I can
tell you this: Try to get a primary conjugated antibody where possible.
The more steps you have, the more steps that can go wrong. It's also
less expensive to get a primary conjugated antibody. Many antibodies
that are made for FACS can also be used for immunofluorescent or

Toshi Akima

On 09/04/2005, at 3:41 AM, Till, Renee wrote:

 > Is there a basic protocol for doing fluorescent IHC on frozen or
 > paraffin sections? Is it better to use a primary antibody conjugated
 > with FITC or whatever, instead of with the secondary? I never thought
 > it> would be such a headache figuring out fluorescence staining on
 > sectioned tissues as opposed to cells. Also, do you need to get all the 
 > separate, or can some of the detections kits be used for fluorescence?

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

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