[Histonet] Correction to Re: Autofluorescence/Imunofluorescence protocols rat kidney
Please excuse the "DITC" mispelling, I meant FITC. I blame it on stumbling
fingers on keyboard!
Just a "DITC" day
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
>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:
>>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!
>>Histonet mailing list
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