Re: [Histonet] Re: Autofluorescence/Imunofluorescence protocols rat kidney

From:Gayle Callis

You will get rid of autofluorescence or most of it by not fixing in an 
aldehyde, this has been discussed recently and many times on Histonet, so 
be sure to search archives for some explanations.   DITC and your 
autofluorescence are pretty much the same color.  If you do snap frozen 
fresh tissue, and another kind of fixation (acetone, acetone/alcohol) you 
will have less problems here.  OR you can try a different fluorophore, i.e. 
a rhodamine RRX from Jackson OR a secondary conjugated to ALexa 555.  Red 
color should come through with autofluorescence as the counterstain, so to 

It is not so important to see the perfect morphology enjoyed by PFA 
fixation, but your IFA staining may improve greatly.  You can use a 
mounting media with DAPI also, this brings in the nuclei as a blue color - 
for contrast and general morphology identification/location of antigen in 

Molecular Probes also has secondaries conjugated to Alexa fluorophores, 
very bright, excellent.

I have a review of autofluorescence I am attaching to you privately, it is 
very informative on fixation, and this problem, etc.

At 05:38 AM 5/16/2006, you wrote:
>Hi Histonet,
>A very basic question but I am a complete novice in the
>world of immunohistochemistry / immunofluorescence and am
>trying to teach myself the rudiments!
>At present, I am attempting to visualise NHE3 in rat kidney
>using immunofluorescence but am encountering a lot of
>autofluorescence.  I just wanted to enquire about possible
>reasons for / methods of dealing with this.
>My method is as follows:
>Once the kidneys have been excised I fix them in 4%
>paraformaldehyde overnight (4 degrees celsius) and then
>incubate them in 30% sucrose until they sink. Kidneys are
>then frozen in isopentane in liquid nitrogen and
>Sections rinsed in distilled water
>3 X 5 min washes in PBS
>Incubation in blocking solution (3% normal goat serum, 0.25%
>Triton X. 96.75% PBS) for 1 hr @ room temp
>Slides then incubated with primary antibody (1:500 chemicon
>anti nhe3) in a humidified chamber @ 4  degrees overnight
>Slides rinsed in distilled water 3 X 5 min washes in PBS
>Incubation with 1:500 FITC conjugated secondary antibody for
>1 hr at room temp
>slides rinsed and coverslips applied with vectashield,
>sealed and visualised
>Apologies for all the detail, just generally unsure of my
>method... and whether there are more apt methodologies for
>this? Getting a lot of autofluorescence and finding it quite
>difficult to visualise the NHE3 at all!
>Would really appreciate any help at all!
>Thank you,
>Histonet mailing list

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

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