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

[Gayle Callis]

Please excuse the "DITC" mispelling, I meant FITC.  I blame it on stumbling 
fingers on keyboard!

Just a "DITC" day

Gayle Callis

At 09:31 AM 5/16/2006, you wrote:
>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 speak.
>
>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 
>tissue.
>
>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
>>cryosectioned.
>>
>>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,
>>Claire
>>
>>
>>
>>
>>
>>
>>
>>
>>
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>
>Gayle Callis
>MT,HT,HTL(ASCP)
>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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