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Author Topic: ICC work but IF doesn't work. Why?  (Read 6481 times)

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Offline apple

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ICC work but IF doesn't work. Why?
« on: January 29, 2006, 03:05:47 PM »
Hi Everybody here,

I did both Immunocytochemistry and immunofluorescent staining of cytospin slides.

ICC worked fine. I can see the dark brown positive signal (used Biotin labelled secondary antibody, avidinHRP, and DAB kit). No dark brown positive color on control slide (no primary ab).

However for IF method. I found positive signals on both control (no primary ab)and sample slides.  I used the same primary ab and Biotin labelled secondary antibody with ICC method and Avidin labelled FITC.  

I fixed all the slides in 4% PFA for 10 minutes. I think this might be the reason. PFA can cause autofluorescence and non specific binding.

Am I right? Is there any other reason?

Thanks

ICC work but IF doesn't work. Why?
« on: January 29, 2006, 03:05:47 PM »

Offline richard03

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ICC work but IF doesn't work. Why?
« Reply #1 on: January 29, 2006, 04:38:43 PM »
Check an unstained slide. If you see positive signal similar to the stained slide, you may have autofluorescence. If you did not see autofluorescence, it may be caused by endogenous biotin so an avidin/biotin blocking step may be needed.

Richard

Offline apple

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ICC work but IF doesn't work. Why?
« Reply #2 on: January 30, 2006, 03:26:20 AM »
I don't think it is because of endogenous Biotin. I didn't block endogenous Biotin with the ICC stained slides either.  But ICC worked fine.

I used exactly the same IF staining method before for another antibody on the same cells. That time, it worked fine. I can see FITC (green) positive color and PI (red) counterstaining color on sample. And only see PI on the no primary ab control slides.

I will try to use acetone fix the slides and do IF again. Because last time I used IF staining a CD marker with colde acetone fixation.

What do you think?

Thanks

Offline richard03

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ICC work but IF doesn't work. Why?
« Reply #3 on: January 30, 2006, 07:44:32 AM »
Then PFA may be the cause. Give it a try and let me know.

Quote from: "apple"


I will try to use acetone fix the slides and do IF again. Because last time I used IF staining a CD marker with colde acetone fixation.

What do you think?

Thanks

Offline ImmunoNYC

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ICC work but IF doesn't work. Why?
« Reply #4 on: January 30, 2006, 11:09:35 PM »
How bright is the signal in the IF slides? Sounds like perhaps you are mistaking autofluorescence or background for real signal.

Since you had success with another antibody, with this protocol on these cells, I recommend you include it as a "reagent positive control" when you do staining with "unknowns".

Also "no primary" is NOT a sufficient negative control. You should do non-immune IgG or better yet an isotype control.
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Offline apple

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ICC work but IF doesn't work. Why?
« Reply #5 on: February 02, 2006, 08:33:40 PM »
hi, I did IF staining today. I fixed the cytospins with cold acetone . I also checked autofluorescence and there isn't autoluorescence at all.
My results show both sample and negative control are negative. No positive color is observed on all the slides.  

But few days ago, I did IF staining PFA fixed cytospins, and i saw positive color on all the slides including negative control.

Also, I did ICC staining of PFA fixed cytospins, and only sample showed positive color (dark brown) and negative control didn't show positive color.  

So ICC and IF staining gave me different results. How to explain these results? What should I do next? Should I try ICC staining of acetone fixed cytospins?

Many thanks

Offline richard03

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ICC work but IF doesn't work. Why?
« Reply #6 on: February 02, 2006, 08:47:34 PM »
I would stay away from IF and concentrate on ICC at this point.

Quote from: "apple"


So ICC and IF staining gave me different results. How to explain these results? What should I do next? Should I try ICC staining of acetone fixed cytospins?

Many thanks

Offline ImmunoNYC

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ICC work but IF doesn't work. Why?
« Reply #7 on: February 02, 2006, 08:47:38 PM »
I have to be honest with you ... you have a lot of threads about the same subject going so I am pretty much entirely confused  ... but that being said .....

From what you said below (see quote) I don't think what you are calling positive is positive. If you see "positive" in your negative controls then it is some sort of BACKGROUND or AUTOFLUORESCENCE or FIXATIVE INDUCED FLUORESCENCE.

I think comparing ICC to IF with different fixatives and different protocols is like comparing apples to oranges.

I guess I would ask what is your ultimate objective? When you answer that, let's talk about protocol details more if you desire.
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Quote from: "apple"
.... and i saw positive color on all the slides including negative control.

Offline apple

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ICC work but IF doesn't work. Why?
« Reply #8 on: February 03, 2006, 05:01:01 AM »
My ultimate objective is:

1. To find out IF or ICC, which method can give me the best result for both cytospin and FFPE sections.
2. The best fixation method for cytospin and FFPE sections.
3. The best protocol for the new antibody.
4. Conclude my samples are positive or negative to this antibody.

I have to work out all of this by myself and within 2 weeks. All of my supervisors don't help me with technique. So I have to turn to here.

Thanks for helping.

ICC work but IF doesn't work. Why?
« Reply #8 on: February 03, 2006, 05:01:01 AM »