A number of things should be remembered and rules followed for obtaining images with colocalization suitable for quantitative analysis:
• 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.
• Use cover glass of the same thickness 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.
• Use plan apochromatic lenses to reduce chromatic shift.
• Use optimized emission filters to maximize emission collection while avoiding bleedthrough.
• Use the same objective lens for the samples you will be comparing.
• Make sure the size of the microscope pinhole is properly set.
• Acquire images sequentially to minimize bleedthrough.
• Acquire images at maximum resolution to ensure reliability of analysis.
• Do not acquire too bright and too contrast images, as it will result in their saturation.
• Do not resave files in any other format than lossless TIFF. It may cause loss of the original image data needed to perform calculations.
• Do not manipulate images in graphics-editing programs, as it may ruin their suitability for quantification.
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