Colocalization guidelines

Sample preparation

• Use specific antibodies that do not cross-react with each other.
• Choose fluorophores with distinct excitation and emission spectra.
• Ensure that the same mounting medium and cover glass thickness are used for all samples being compared.
• Avoid using anti-fading reagents, as these can increase background fluorescence.
• Include unstained control samples to assess autofluorescence.

Microscope set-up

• Utilize plan apochromatic lenses to minimize chromatic shift.
• Employ optimized emission filters to maximize the collection of emissions while minimizing bleedthrough.
• Use the same objective lens for the samples being compared.
• Ensure that the size of the microscope pinhole is set correctly.

Image acquisition and handling

• Acquire images sequentially to reduce bleedthrough.
• Capture images at the highest possible resolution to ensure reliable results.
• Avoid capturing excessively bright and high-contrast images, as these may become saturated.
• Do not resave files in any format other than lossless TIFF, as this can  prevent loss of original image data.
• Avoid using JPEG format for image files.
• Refrain from manipulating images in graphic-editing programs, as this may affect their suitability for quantification.
• Always retain the original files, as they may be needed for reference  to the original software used for acquisition and to confirm image metadata.