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Author Topic: Help Needed with the following Image  (Read 6795 times)

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Offline ghostdog

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Help Needed with the following Image
« on: August 19, 2008, 11:42:31 AM »
http://www.ihcworld.com/imagegallery/displayimage.php?album=16&pos=0

Spleen from a luciferase+ve mouse stained with an anti-luciferase ab.
I would love to know what the very strong "dotty" staining in the spleen is. Its not present on control sections lacking antibody.


thanks


Help Needed with the following Image
« on: August 19, 2008, 11:42:31 AM »

Offline ghostdog

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Re: Help Needed with the following Image
« Reply #1 on: August 28, 2008, 05:49:54 AM »
anyone? :(

Offline richard03

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Re: Help Needed with the following Image
« Reply #2 on: August 30, 2008, 04:08:41 PM »
Could be due to primary antibody. Try more diluted primary antibody.

Offline ghostdog

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Re: Help Needed with the following Image
« Reply #3 on: September 01, 2008, 05:25:30 AM »
Hi Richard

thanks for your reply, i should have stated earleir, i have performed dilutions from 1:50 all the way out to 1:1000. They "spots" are still there.


Offline CanuckPhD

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Re: Help Needed with the following Image
« Reply #4 on: September 01, 2008, 05:52:03 AM »
How did you fix the tissue? How is the luciferase expression being driven (that is what is the promoter for it)?

Offline ghostdog

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Re: Help Needed with the following Image
« Reply #5 on: September 01, 2008, 09:04:44 AM »
tissue was fixed in ice  cold acetone, the luciferase is based on a beta actin reporter.

Offline richard03

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Re: Help Needed with the following Image
« Reply #6 on: September 01, 2008, 10:19:48 AM »
Abcam has a Luciferase antibody. PFA seems to be the optimal fixation method. But I also question the purity of your antibody.

http://www.abcam.com/index.html?datasheet=21176

Offline ghostdog

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Re: Help Needed with the following Image
« Reply #7 on: September 01, 2008, 10:33:16 AM »
Hi Richard

the antibody i am using is a Promega one, when you say purity do you mean it looks like the antibody has been contaminated or do you mean it is just not very selective?

Offline richard03

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Re: Help Needed with the following Image
« Reply #8 on: September 01, 2008, 10:55:43 AM »
Since you have already tried 1:1000 dilution, I would then think that the antibody may not be very selective. Usually contamination issue can be solved by using highly diluted antibody. If you want you can try more dilution range (i.e. 1:2000, 1:5000).

 

Offline ghostdog

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Re: Help Needed with the following Image
« Reply #9 on: September 01, 2008, 01:16:17 PM »
Hi Richard
thanks for this, the original dilution suggested was one in 50 and i did think of impurity, so that when i went out to 1:1000. I will try it and also i will try a non fluoorescent approach. See if that gives anymore leads"

Offline ghostdog

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Re: Help Needed with the following Image
« Reply #10 on: September 03, 2008, 04:59:02 AM »
to follow up, i stained at 1:2000 and 1:5000, and still the same problem.  :(

Offline CanuckPhD

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Re: Help Needed with the following Image
« Reply #11 on: September 03, 2008, 08:49:26 AM »
What does the tissue look like with an H&E? From the looks of the picture you may not have fixed the tissue well and got lysis of the cells when you performed the staining. This may account for some of your staining pattern. As well what does goat IgG as a negative control look like at the same concentration?

Also have you looked at any other tissues in the mouse to see if this staining is found only in the spleen? I noticed that this antibody is recommended for immunocytochemistry. Have you tried it on cytospins of splenocytes to ensure that it works?

Just throwing some ideas out there. Good luck.

Offline ghostdog

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Re: Help Needed with the following Image
« Reply #12 on: September 04, 2008, 06:20:31 AM »
Thanks for the suggestions Canuck, the igG controls are clear, no dotty staining at all. i have not done h+E yet but will be shortly, i am also going to try and do a non-fluorescence based stain.

I snap froze the tissue, i think that this may not have helped either but i am not certain of that either.

Re: Help Needed with the following Image
« Reply #12 on: September 04, 2008, 06:20:31 AM »