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Author Topic: non specific nuclear staining in IF  (Read 7459 times)

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Offline kyong

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non specific nuclear staining in IF
« on: February 02, 2005, 05:47:06 PM »
Hi!

Does any body know why?

I used mouse kidney frozen section, primary Ab is Sheep anti human CAII, and secondary Ab is donkey anti sheep Alexa AF488, we used the same primary Ab and DAB method and it worked well, that means the primary Ab is working in mouse tissue, but when I used IF method, I can see nuclear staining also.

Thank you for your time!
im
Renal Lab, Medicine Dept.,
Univ. of Arizona

non specific nuclear staining in IF
« on: February 02, 2005, 05:47:06 PM »

Offline richard03

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non specific nuclear staining in IF
« Reply #1 on: February 03, 2005, 09:07:52 PM »
That was probably autofluorescence background. The nuclear staining may be blood cells. Check a unstained slide to see if you can observe the same nuclear staining.

Richard

Offline kyong

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non specific nuclear staining in IF
« Reply #2 on: February 04, 2005, 12:06:53 PM »
Hi!Richard,

Thank you for your reply!

I am sure those nuclear staining are not autofluorescent. Only few vessel in mouse kidney will have autofluorescent, but not nucleus.
im
Renal Lab, Medicine Dept.,
Univ. of Arizona

Offline richard03

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non specific nuclear staining in IF
« Reply #3 on: February 04, 2005, 10:19:59 PM »
Can you post an image in the gallery? Since some blood cell staining will look like nucleus.

Offline kyong

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Re: non specific nuclear staining in IF
« Reply #4 on: February 09, 2005, 01:05:19 PM »
Hi! Richard,

Thanks again!
I don't know how to post the image!
im
Renal Lab, Medicine Dept.,
Univ. of Arizona

Offline richard03

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non specific nuclear staining in IF
« Reply #5 on: February 10, 2005, 11:20:56 PM »
Go to image gallery (http://www.ihcworld.com/imagegallery/). Click on Register and this will guide you through the registration. Once register, go back to image gallery and Login using the user name and password you selected. The click on upload and select the image file from your local computer and upload the file.

Offline kyong

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non specific nuclear staining in IF
« Reply #6 on: February 16, 2005, 06:51:56 PM »
Hi!Richard,

I had already posted the image, please view it at the gallery.
im
Renal Lab, Medicine Dept.,
Univ. of Arizona

Offline richard03

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non specific nuclear staining in IF
« Reply #7 on: February 16, 2005, 10:36:08 PM »
Kim,

I looked at the image and it is indeed nuclear staining of kidney cells (not blood cells).  Looks very specific to the nuclei.

As you have already used DAB method and it worked well, so I think the problem may come from the donkey anti sheep Alexa AF488. You can do a test to incubate the sections with this secondary ab ONLY. See what will happen. Let me know.

Richard

Offline newtmo

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Re: non specific nuclear staining in IF
« Reply #8 on: March 08, 2010, 01:51:23 PM »
I've had good luck quenching this non-specific (or cross reactive?) type of stain by including 1-5% Bovine Serum Albumin in my blocking step.  It only seems to occur with certain primary ABs.

Re: non specific nuclear staining in IF
« Reply #8 on: March 08, 2010, 01:51:23 PM »