Thanks Rachel

But should i select the desired area after thresholding or directly without the need to perform thresold for my image??

Depending of your cell number outline control step by step each cell and use histogram to see and save distribution of signal level. Repeat operation for cells of interest. 

COmpare data from control and experimental cells.

You can use any method suitable to make selection. But you will colect more data from individual selection.

No, i'm not currently working with immunofluorescence ... just immunohistochemically stained tissue sections.

Thank you soooo much

Indeed, you shouldn't measure intensity in a thresholded image.

You could use a thresholded image to create masks or regions of interest (ROIs), which you then can use on your original data. This saves you the trouble of manually selecting your ROIs.

But if you only have a few images manual selection is probably faster.

Thank you Tom ..

But would you, kindly, clarify how can i specify regions of interest in thresholded images & then calculate staining intensity in it?

In brief, if you have a (quasi) noise-free thresholded image, you can use 'Analyze' -> 'Analyze Particles ...'. In the interface of this this command you can select the size-range of particles that you want to select as ROIs. If you know the scale in you image you can use microns, otherwise just use pixel units. You can also set a selection range based on circularity of the particles.

The most important thing however, to get the ROIs into the ROI Manager, is to select 'Add to Manager'. Then just click 'OK'.

Remark: This procedure works well for particles (objects of interest) that are spatially clearly divided in your image. If you have particles that are touching each other, you could first apply 'Process' -> 'Binary' -> 'Watershed' to try to split these particles. And only then continue with 'Analyze' -> 'Analyze Particles'.

Once your ROIs are defined in the ROI Manager, you can use them in your original image by selecting this image and then selecting a specific ROI from the ROI Manager. To measure properties using a specific ROI, you first have to select which properties you want to measure. You can do this in 'Analyze' -> 'Set Measurements'. Once selected, you can measure these properties by selecting an image, selecting a ROI and pressing 'Measure', directly in the ROI Manager. To speed up this process you can measure all ROIs at the same time by selecting them all (ctr+a or cmd+a) and then pressing 'Measure'.

Hope this helps, good luck!

Tom