Hi there Carla Amado ,

1- However, I am aware that depending on the tissue preparation including fixing, staining, munting etc & imaging process such as in/outplane fluorescence, configuration may affect pixel intensity?

It seems you are facing the mean gray value vs integrated density conundrum. About the differences of these two parameters we have discussed extensively in RG:

(https://www.researchgate.net/post/Mean_gray_intensity_value_or_the_integrated_density_value_which_among_the_two_value_is_more_appropriate_for_quantifying_the_fluorescence_image_data)

And also at the IJ forum:

(https://forum.image.sc/t/mean-grey-value-or-integrated-density/82474)

Out of plane fluorescence shouldn't be an issue when working with a confocal microscope (as long as you take care of the pinhole) and every effort should be made to equalize acquisition conditions, etc.

Is there a way I can just count the pixels in the region of interest? and compare how many this is so that i can quantify protein?

The problem is how do you define your region of interest. If you threshold the images, then fluorescence values will affect the threshold you choose. If you are trying to measure quantity of a protein, it is a good practice to measure intensities, not just areas. You can express things as integrated densities, which is a balance between the quantity of fluorescence and the area.

I am just concerned pixel value will alter due to experimental minor differences that it wont be comparable between experimental group & controls

This is why you should run experiments in parallel. Any day you perform an experiment you should have a control in it (Treatment vs non treatment, WT vs KO, ...). That way you'll be able to always compare to your inner control, and then pool the data by normalizing to your control values.

I agree the differences in fluorescent staining between days can be huge (and due to many possible reasons, fixation, antibodies, washes...).

You probably cannot compare treatment of day2 with control of day1, but you should be able to compare treatment of day2 to control of day2. Then you can normalize to Control values and pool your data from different days together.

2. Also does changing brightness/contrast on zeiss whilst imaging (this is used to monitor saturation) is it also changing pixel value? This is because diff images require diff brightness/contrast in order to see the fluorescence & reduce saturation?

Usually, the display doesn't change the raw values. If you don't change parameters as the power gain, the offset, the range of the photomultipliers, etc, the raw values of the images are not affected by changing the display values. If it is ZEN software, the display doesn't affect the raw values at all.

I hope this helps,

Cheers,

J