Author Topic: immunohistochemistry and western blot  (Read 15533 times)

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exile

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immunohistochemistry and western blot
« on: September 15, 2006, 12:45:19 PM »
I have done IHC and WB experiments on retina with the same animal model. There was no obvious change in IHC but a mild change in WB which showed significant difference in statistics.
How to explain this?

immunohistochemistry and western blot
« on: September 15, 2006, 12:45:19 PM »

Offline excalibur

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immunohistochemistry and western blot
« Reply #1 on: September 15, 2006, 04:47:35 PM »
Western blots are quantitative and more sensitive.
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Offline ImmunoNYC

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immunohistochemistry and western blot
« Reply #2 on: September 30, 2006, 01:56:06 AM »
Westerns are semi-quantitative and I agree that they are MUCH more sensitive. IHC can be pseudo-semi-quantitative however you must be careful as often times people over-develop their slides and are no longer in a linear range of enzymatic activity and subtle differences between staining intensities may be lost.

Also westerns applications call for processing proteins very differently than for IHC so perhaps there are subtle differences in the antigen binding sites between the two processes that give you the discrepant results. Are you using the same primary?

If you truly want to find out if you have a difference you may want to go to a truly quantitative method such as ELISA etc.
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exile

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immunohistochemistry and western blot
« Reply #3 on: September 30, 2006, 02:08:43 AM »
yes, I used the same primary antibidy .
Now I am worry about how to write the paper. Maybe some editors will doubt the result.

Offline ImmunoNYC

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immunohistochemistry and western blot
« Reply #4 on: September 30, 2006, 02:13:54 AM »
Are your results in the western blots reproducible over at least 3 experiments?? If so, I would trust the western data more. Do you have other assays you can confirm your results. You can show the IHC data in the supplemental data or as data not shown and just mention you saw no differences but attribute it to lack of quantitative abilities in IHC. If you do immunofluorescence - and perhaps do not use an enzymatically amplified system and do not use avidin-biotin - the immunos may turn out to be more quantitative.

Good luck!
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Quote from: "exile"
yes, I used the same primary antibidy .
Now I am worry about how to write the paper. Maybe some editors will doubt the result.

exile

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immunohistochemistry and western blot
« Reply #5 on: September 30, 2006, 03:15:36 AM »
Of course I have done WB at least for three time.
As you said, I used immunofluorescence in IHC and maybe this is one of the reason.
 I will write the paper as you said.Thank you very much!

Offline ImmunoNYC

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immunohistochemistry and western blot
« Reply #6 on: October 01, 2006, 01:22:46 AM »
Since westerns are reproducible x3 as you said, more sensitive, and more quantitative - I would believe those results.

However, since you used immunofluorescence I would expect it to be more quantitative than IHC based on an enzymatic reaction such as DAB development, but again there are so many variables in staining, like making sure ALL slides are treated EXACTLY the same, that technical variability is expected. Westerns are done on one blot only with all groups together one one membrane so the variation is significantly less between treatment groups.  

Good luck and let us know what the reviewers say. I am curious to know now.
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Quote from: "exile"
Of course I have done WB at least for three time.
As you said, I used immunofluorescence in IHC and maybe this is one of the reason.
 I will write the paper as you said.Thank you very much!

immunohistochemistry and western blot
« Reply #6 on: October 01, 2006, 01:22:46 AM »