Posted by
Sean Burke on
Sep 12, 2012; 8:11pm
URL: http://imagej.273.s1.nabble.com/Fluorescence-intensity-analysis-with-tissues-tp5000037p5000042.html
Hi Sebastian,
Have you thought about exporting to FCS Express Image Cytometry for analysis of the segmented data. There are some people doing this with histology sections already and there are some examples at
https://www.denovosoftware.com/site/ImageApplicationExamples.shtml#histology.
Regards,
Sean Burke
-----Original Message-----
From: ImageJ Interest Group [mailto:
[hidden email]] On Behalf Of spetry
Sent: Wednesday, September 12, 2012 2:13 PM
To:
[hidden email]
Subject: Fluorescence intensity analysis with tissues
Hello there,
the quantative analysis of immunostained specimen troubles me and unfortunately the few things which I could find on google and in this forum did not match my concerns, so I deceided to open a new thread and ask for help.
I am working with a mouse model and got PCR results from isolating islets of Langerhans from the pancreatica. Now I want to confirm the results with immunofluorescence, to be precise: intensity measurement of target proteins on mouse pancreatic tissue, amongst which insulin.
Here is my method:
I started by taking pictures of the islets on my slides and used exactly the same settings (time, gain, gamma, ...). The same was done with blank slides (without primary antibody).
In Image J I used the image calculator to substract the picture from the blank slide from the picture I want to analyse in order to remove background.
Then I selected the islet area with the freehand tool and measured the mean grey value.
Now here are my questions:
1) Can you do it like this or did I miss something?
2) I read that people normalize the intensity with the area when working with cells. Should you also do that when working with tissue and if yes, how is the best way to do it?
3) What about using a threshold in order to get clearer results? I thought about it, but I got pictures with very low staining and others which very high intensity, so I could not use the same threshold for all pictures. I wonder whether it's a good idea or even scientifically correct to set the threshold to the mean grey value +/- 50 % on every picture or something like that.
Maybe someone can give me some inspiration, thank you in advance! :)
Best regards,
Sebastian
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