Re: Fluorescence intensity analysis with tissues

Posted by spetry on Sep 14, 2012; 11:38pm
URL: http://imagej.273.s1.nabble.com/Fluorescence-intensity-analysis-with-tissues-tp5000037p5000087.html

Hello,

I followed the idea of using nuclei stained with Hoechst 33342 as an internal control. Hoechst does not need secondary antibodies, it's a fluorochrome itself.
As mentioned above I've got slides without any primary antibody, two per timepoint and animal which means 36 samples in total (remember: three animals per timepoint, three timepoints, two groups of mice).

I took two pictures of random locations from each sample and calculated the average for every animal and timepoint which makes 18 average intensity values.
Then, I calculated the common average in order to see how much the staining intensities of the nuclei stained with Hoechst 33342 vary from it on each animal.

The most extreme average values are 5 % away from the total average, so I suppose that I can use Hoechst as the internal control.


Now I am thinking of the following in order to normalize the intensities of my target proteins:

Again, I will take pictures from nuclei stained with Hoechst, this time from the slides which were stained with the respective protein, separately for each protein. This way, only batches will be compared. I will make Hoechst intensity averages for each animal and normalize those by the total average of all animals in this batch.


Now I've got two questions:

1) Is my conclusion itself about the low variation in nucleus staining intensity as well as the method with which it was achieved (using slides w/o primary antibody) reasonable and correct?

2) Are you allowed to normalize the staining intensity the way I suggest (normalizing a protein in a batch to this batch's own nucleus intensity average)?

Thank you in advance for replies!

Best regards,

Sebastian