A number of things should be remembered and rules followed for obtaining confocal images with colocalization suitable for quantitative analysis:

Sample preparation:

• Make sure that the used antibodies are specific and do not cross-react.

• Choose fluorophores with well-separated excitation and emission spectra.

• Use the same mounting medium for the samples you will be comparing.

• Try to avoid using anti-fading reagents, as they can increase background fluorescence.

• Use unstained control samples to check for autofluorescence.

Microscope set-up:

• Use plan apochromatic lenses to reduce chromatic shift.

• Use optimized emission filters to maximize emission collection while avoiding bleed-through effect.

• Use the same objective lens for the samples you will be comparing.

• Make sure that the size of microscope pinhole is properly set.

Image acquisition and handling:

• Acquire images sequentially to minimize bleed-through effect.

• Do not acquire too bright and too contrast images, as it will result in their saturation.

• Do not manipulate images in graphics-editing programs, as it may ruin usability of your images for quantitation.

• Do not resave files in any other format than TIFF. It may cause loss of the original image data needed to perform calculations.

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