Immunofluorescent staining with Alexa dyes

From:Gayle Callis

We use Strepavidin Alexa fluorochromes a great deal for IFA on sections and
never add Tween 20 to buffers.  Instead, we use normal serums or Jackson
Immunoresearch BSA (protease and immunoglobulin free) to buffers until the
Alexa step as normal serum blocks, dilutents of biotinylated primary or
primary, then biotin-secondary. If I remember correctly and also via
private communication with Molecular Probes technical services, they do not
recommend addiion of proteins or detergents with Alexa dyes.  We thought to
try it but opted for their recommendation to use pure buffer for any Alexa
dye steps.   The sections before Alexa step are rinsed with pure buffer to
get rid of any proteins, then Alexa's diluted in pure buffer (we tend to
use a large volume over section). For sections our Strepavidin Alexa 488 or
546 was diluted 1:1000 per their recommendation, but if you don't protect
the dye from light or leave it out over a period of time that dilution
works towards 1:200.  

One thing to keep in mind, once Alexa fluorochrome conjugates are diluted,
we found the one year expiration date very important, and be sure to
CAREFULLY protect these dyes from light in storage (use black boxes) and
during staining. It is a good idea to aliquot and freeze down at -27C to
maintain good reagent.  We noticed some loss of fluorescence over time with
single aliquots that were exposed to light many times or sitting not being
used for several weeks, and to insure the perfect experiment (when in
doubt!) a new aliquot was used. 

These dyes are wonderful, and have replaced FITC and rhodamine in most of
our IFA procedures, even for FACS, due to their resistance to quenching
with exciting UV light. 

Due to Alexas brightness, PFA fixation worked well. We did a PFA
concentrations and time study, 1,2,3,4% versus each conc/time, 1,2,3,4,5,
etc) to find optimal fixation for some murine CD markers or Salmonella sp
IFA. We prefer to use acetone or acetone/alcohol fixed frozen section, but
your markers could work well with other fixation. 

You wrote: 

I am working on a protocol for multiple immunofluorescence labelling on
frozen sections using Alexa fluors 488, 546 and 647 and I need some
information for the following questions:

1) Will the addition of Tween-20 to the washing PBS have any damaging
effect on the fluorochromes in terms of specific binding to the markers
concerned as well as on the subsequent stability of the fluorescence in
the final preparation?
2) My markers of interest are anti-Thioredoxin, anti-APE/Ref-1 and
anti-CA IX.
Is paraformaldehyde/formaldehyde the best choice of fixative for these

Any feedback on these questions or any well-established protocol on

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367 (voice mail)
406 994-4303 (FAX)


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