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Depending on the target of interest, direct or indirect visualization is recommended.
Direct visualization requires the use of a labeled primary antibody:
Short protocol, fast analysis, easy to handle.
Expensive, a labeled primary antibody for each target is needed.
A highly specific primary antibody is needed.
No signal amplification, not suitable for low expressed targets.
Indirect visualization requires the use of a labeled secondary antibody:
Less expensive, a labeled secondary antibody can be used for different primary antibodies.
Higher background noise.
Signal amplification, suitable for medium to high expressed targets.
|Conventional fluorescent labels||Medium bright and photostable, replicate historic experiments||FITC, R-PE, TRITC, Cy3|
|Alexa® labels, DyLight||Cover the white range, common filter sets, bright, photostable, not pH sensitive||Covering different wavelengths|
|QDots||Single light source, multiplex, narrow emission||Covering different wavelengths|
Since a fluorescent signal fades away during time, mounting media is added at the end of the staining protocol to maintain the sample. It is also needed to optimize the refractive index for imaging. Mounting media are a solution of glycerol in a special buffer. This helps to maintain the fluorescence signal and slow down photobleaching. Mounting media are optimized for different conditions and dyes, and some questions to consider when selecting the right one are:
Fixed samples for long-term conservation?
Fixed samples for immediate imaging?
Which kind of dye/fluorophore?
|Tip: Poorly prepared samples show artifacts that could be misinterpreted. For instance, bubbles, insufficient washing, over-incubation with antibodies, or over-fixation can result in unspecific staining patterns and show false-positive results. Adding too much or too little mounting media can create artifacts and damage the sample.|