I have performed ISH on slides of post mortem brain tissue (region BA46, DLPFC) with 35S-UTP labelled riboprobe. I then dipped the slides in Kodak emulsion, developed with a D19 developer and counterstained with Nissl.
I am interested in a high throughput method to quantify the silver grains, so I can report it as "grains/cell" and/or "density of label/cell".
I looked at Aperio Imagescope and BD Pathway 855 Bioimager. Does anyone have experience with these systems and softwares? Are they trustworthy when compared to manual methods using steroinvestigator/adobe photoshop/Image J (the tried and tested hard manual labour methods?!). Does anyone have a protocol floating around that I can look at?
if I understand correctly, you are interested in counting the number of "grains" in the background per total background, and compare this to number of cells in the image?
would want to look at an image to see what exactly describing.
If I understand correctly need to set a threshold for pixel intensity and call that a "grain", and compare to total background pixels. Have done something somewhat similar with images using the imaging toolbox in MATLAB.
thank you
Thanks Edmund. I have attached a 20x image. What I actually interested in is the 'grain count/cell' (where cells will be pyramidal or interneurons) and 'density of label/cell' (which I would expect to report for example like this - "Pyramidal neurons in layer 3 of the cortex expressed more mRNA of protein X when compared to interneurons or pyramidal neurons in all other layers").
The grains are the actual silver grains that are really the brownish change in the silver molecules in the Kodak emulsion due to the radioactivity given out by the 35S-UTP labelled riboprobe: so I am looking at a way to quantify the amount of silver grains (ergo desity of the protein mRNA of interest) per cell.
I don't quite understand what you are calling a "cell". Is a "cell" each individual "grain"...many cells in the image field, or is the entrie image covering a single cell"
I think you want to know the density of each "grain" in the field of view? ...the field of view is one cell..?
I would consider looking at the MATLAB Image Processing Toolbox for suggestions.
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