Author Topic: Negative control is positive for IF staining  (Read 10816 times)

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Offline Koi

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Negative control is positive for IF staining
« on: December 06, 2010, 09:25:37 AM »
Hi,

I have been getting positive staining for my negative control (by omitting primary antibody or replacing primary antibody with normal serum).
I am using indirect immunofluorescence technique trying to stain tissue sections of turkey brain. The tissues was fixed with 4% paraformaldehyde.
For negative control sections, I omitted primary antibody but still get the staining from the secondary antibodies. I used Donkey anti-rabbit FITC, Donkey anti-goat FITC from JacksonImmunoResearch and Goat anti-rabbit FITC, Donkey anti-goat FITC from Santacruz. I blocked the sections with normal donkey serum in the first step (tried both 5% and 10% for 30 min with and without BSA). I incubated secondary antibody for 1 hr in dark. Does anyone know what can possibly cause positive staining when there is no primary antibody? I called it positive staining because it is not the background staining and it stains only on the specific area that I am interested in. I know there are many types of cells surrounded in the tissue section since I use the whole brain sections. But, all those cells in different areas did not show any staining.
There is no autofluorescence in that area.

Any ideas would be appreciated.

Thanks,
Koi

Negative control is positive for IF staining
« on: December 06, 2010, 09:25:37 AM »

Offline Nick101

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Re: Negative control is positive for IF staining
« Reply #1 on: December 06, 2010, 01:39:17 PM »
I have never had that happen. However I can only suggest that you double check each step of the staining. Is it possible you grabbed the wrong slide? Is it possible your hydrophobic pen is not holding the solutions in place and leaking is occurring (this one depends on your method of staining)?

Offline Cardio

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Re: Negative control is positive for IF staining
« Reply #2 on: December 06, 2010, 02:03:31 PM »
DId all 4 secondaries give you the same positive staining or was it just one?

Could you post a picture of both primary/secondary and your negative control.

Offline Koi

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Re: Negative control is positive for IF staining
« Reply #3 on: December 06, 2010, 03:11:09 PM »
Yes, all 4 secondary antibodies gave the same staining. Both primary/secondary and secondary alone had the same staining as shown in the picture. I attached only one picture since there are no different in staining pattern/intensity for both of them.

I have only one section/slide and repeated the staining many times to make sure that I did not mess up with something.




Offline Cardio

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Re: Negative control is positive for IF staining
« Reply #4 on: December 07, 2010, 05:42:17 PM »
THe only explanation I have (and its a weak one) is that your secondaries are contiminated with primary. Its probably not that but there is no explanation for the staining you are getting if the autoflouresence is low on the slides without any antibody. You can have secondaries interacting with species IgG but I wouldn't expect all 4 to do it in the same pattern.

Offline Nick101

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Re: Negative control is positive for IF staining
« Reply #5 on: December 07, 2010, 05:44:58 PM »
I would have to agree with Cardio on that one. It sounds like possible contamination. It's hard to think of alternatives as we cannot know every detail of your method and how you carry it out.

Offline Koi

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Re: Negative control is positive for IF staining
« Reply #6 on: December 07, 2010, 09:55:26 PM »
Thanks for the ideas! I am testing the specificity of the secondary antibody by further dilute it to very high dilutions. We have been working on this area with many antibodies and never had autofluorescence issue but I will check it again. 

Offline Koi

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Re: Negative control is positive for IF staining
« Reply #7 on: March 01, 2011, 12:22:06 PM »
Hi,

I just want to let you guys know what went wrong with my staining.
I tried new aliquots of antibodies, new tissue sections, all new reagents... did not solve the problem.
I diluted secondary antibodies further... got the same results.
I omitted secondary antibodies... got beautiful signals!
There is only one possibility left... autofluorescence!
I checked unstained sections. I saw the neurons glowing but the pattern looks so much different from what I got after processing.
The signals looked so real after staining instead of shining brightly and cluster as big spots. I still don't know the reason that could change the staining pattern.
We have been working with this area almost 10 years with no problems. I tried to use autofluorescence eliminating reagent to get rid of autofluorescence but still do not like what I see. Now, I just have to wait until we get young animals and try again.

Thanks a lot for the ideas.

 

Offline pandanac

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Re: Negative control is positive for IF staining
« Reply #8 on: March 12, 2011, 08:05:04 AM »
Hi Guys:

I have had the same problem stated as Koi, I would like to explain mine..I am looking to map the C-fos in the V1 of the rat brain.

1. I use Brain tissue slices, PFA perfused, Fixed, sectioned and mounted on slides and frozen until further use.

2. Used TBS-twn-20 for washing steps.

3. Nonspecific blocking for 2hrs by serum (10%).

4. Primary antibody I used first was from Santa Cruz Dilution 1:100 overnight incubation

5. Secondary antibody Alexa Fluor-594 dilution 1:300 incubation for 2 hrs at room temperature

6. Washed several times and add the DAPI incubated for 20 min

7. Washed, added the antifade and cover slipped.

the problem is I am seeing positive neurons (mean the antibody is binding to neurons which can be seen in the control tissue, which is from the rat not exposed to any visual stimulation [that's what i do to induce the c-fos in the V1 area f the brain ])

I changed the antibody from santa cruz to ABcam antibody, however I am still seeing the same result :-(

can any one help me in solving this problem.

Offline Koi

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Re: Negative control is positive for IF staining
« Reply #9 on: March 12, 2011, 01:14:33 PM »
Hey Pandanac,

I think you should try validating your primary antibodies first. You may get c-fos expression from any kinds of stimulation. What you have got may be real expression at the basal level. Take unstained tissues and check for autofluorescence. Compare control with treated animals is another way to try. If what you got from control sections is not that bad can you set it as the background level when comparing with treated ones?


 

Offline pandanac

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Re: Negative control is positive for IF staining
« Reply #10 on: March 12, 2011, 01:22:23 PM »
Hey!!
thanks for the info.. i will try the suggestions..
will get back to you very soon


thank you so much..

Offline AAdrienne

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Re: Negative control is positive for IF staining
« Reply #11 on: December 08, 2016, 11:56:39 AM »
Is anyone still following this thread? I'm having major problems with positive staining in my negative control. If anyone is interested, I can explain my procedure. Thanks!

Re: Negative control is positive for IF staining
« Reply #11 on: December 08, 2016, 11:56:39 AM »