Weak Or No Signal |
|
Possible Cause |
Recommendations |
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Inappropriate storage of samples; signal may fade if fluorophores are exposed to light for extended periods of time |
Perform incubations and store samples in the dark. Samples may be mounted in an anti-fade solution, such as ProLong® Gold Antifade Reagent #9071. Samples should be imaged immediately following mounting for the best results. |
|
Cell/tissue samples stored for too long |
Use freshly prepared slides/plates to avoid loss of antigenicity. |
|
Inadequate fixation |
Consult the CST product datasheet for the recommended protocol; remove media and quickly/thoroughly wash in fixative immediately after treatment. For phospho-specific antibodies, use at least 4% formaldehyde to inhibit endogenous phosphatases. |
|
Incorrect antibody dilution (antibody too dilute) |
Consult the CST product datasheet or cellsignal.com for the recommended antibody dilution. |
|
Not using the recommended incubation time |
Primary antibody incubation according to a rigorously tested protocol provides consistent, reliable results. CST antibodies have been developed and validated for optimal results when incubated at 4°C overnight. |
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Inappropriate testing model |
If possible, protein expression should be confirmed by western blot or other means. |
|
Target protein not induced properly |
Optimal treatment conditions and controls should be determined for each target. |
|
Low expression of protein of interest |
Modify detection approach; consider signal amplification or pair with a brighter fluorophore. |
|
Wrong permeabilization method |
Consult datasheet for recommended protocol. |
|
Incorrect use of secondary antibody |
Use recommended concentration and check secondary antibody is matched to host species of the primary antibody. |
|
Wrong excitation wavelength |
Ensure illumination and detection (laser/excitation/emission filter) matches excitation wavelength of fluorophore(s). |
|
Low signal in multiplexed IHC |
Optimization of deparaffination, antigen retrieval, and signal amplification methods. |
High Background |
|
Possible Cause |
Recommendations |
|
Sample autofluorescence |
Use unstained samples as controls to check autofluorescence levels. Check datasheet for correct fixation reagent. Old fixative may autofluoresce; replace old formaldehyde stocks and prepare fresh dilutions. Use EM-grade glutaraldehyde freshly diluted from ampules. Choose longer wavelength channels for low-abundance targets. |
|
Insufficient blocking |
Use normal serum from the same species as the secondary antibody used. Consider a charge-based blocker, such as Image-iT® FX Signal Enhancer #11932, depending on the source of background. |
|
Incorrect antibody dilution (primary or secondary antibody too concentrated) |
Consult the CST product datasheet or cellsignal.com for the recommended antibody dilution. |
|
Samples dried out |
It is vital that sample remains covered in liquid throughout the staining procedure. |
|
Insufficient washing |
Wash to remove excess fixative, excess secondary antibody, and loosely bound, non-specific antibody interactions. |
|
Secondary cross-reactivity |
Use isotype control antibodies to determine whether your secondary antibody is cross-reacting. |
|
Non-specific antibody binding |
If available, compare to knockdown (siRNA) or knockout cells, or compare to cells known to express higher or lower levels of the target antigen. |
