Re: Fluorescence intensity analysis with tissues

Posted by Mbar on Sep 18, 2012; 9:06am
URL: http://imagej.273.s1.nabble.com/Fluorescence-intensity-analysis-with-tissues-tp5000037p5000102.html

Hello Sebastian

I followed this tread with great interest, because I also want to measure fluorescence intensities in tissue. I´m staining cell nuclei with DAPI, I think its similar to Hoechst, as DNA stain.
I don’t know, If I get you right: you want no normalize the intensity of your target protein to the average intensity of your nuclear staining?
If I got you right, then I´m unsure with the idea to get an internal control by creating the average of nuclear stains.´, because I made the  experience that immunohistochemistry can go wrong but DAPI staining still works on the specimen- independent of e.g.  age or pretreatment of the specimen.
For example, I treated specimens that were 40jears old-DAPI works great- others didn´t work.  So I think there´s no correlation between DAPI staining and other immunofluorescent signals- and I think for Hoechst it’s the same thing. In my opinion it gives you no more information about your intensities, if you normalize it to nuclear staining intensity.
But the question for me is, if you really need an internal control, I mean are your staining results per sample are so extremely different?
If its like that, perhaps you can find a protein, a typical Langerhans cell marker, then double-stain with your target protein and normalize it to the average intensity of this protein per Langerhans cell?


Best regards,
MBar