Difficult problem 2
Possible cause of high background
- Sample spontaneous fluorescence
Suggestion: Use undyed samples as control to detect the degree of spontaneous fluorescence. Replace the new formaldehyde fixator, the old fixator may produce spontaneous fluorescence; For low abundance targets, longer wavelength channels are selected.
- Inadequate closure
Suggestion: Use normal serum from the same species as the secondary antibody, extend the sealing time appropriately, 4℃ overnight is the best sealing condition.
- Improper use of antibodies, high concentration of primary or secondary antibodies or long incubation time
Suggestion: Use the recommended antibody dilution, the incubation time of the antibody is not too long. The primary antibody can be closed overnight at 4℃, and the incubation time of the second antibody can be 1-3 hours.
- Sample dries
Suggestion: Keep the sample in liquid at all times throughout the dyeing process to avoid drying out the sample.
- Not cleaning enough
Suggestion: Full cleaning to remove residual fixatives, antibodies and other non-specific binding effects.
- Cross-reactivity of secondary antibodies
Suggestion: Use homologous control antibodies to determine whether the secondary antibody is cross-reactive.
- The non-specific binding of the second antibody itself is serious
Suggestion: It is suggested to set a negative control, do not incubate the primary antibody, and directly incubate the secondary antibody in the sample to observe the non-specific binding. If there is a nonspecific binding, it is recommended to replace the secondary antibody.
Abbkine IF kit, equipped with all reagent components required by IF, optimized experiment scheme, make your IF easier and more convenient.
Advantages and characteristics
Convenient operation, complete components, do not need to worry about complex reagents, open the box can start the experiment.
Provide optimized antibody diluent and SuperKine™ enhanced anti-fluorescence quencher for strong anti-quenching and excellent fluorescence intensity.
Dylight series of secondary antibodies, with stronger fluorescence intensity and higher optical tolerance and specificity.
Provide the serum from the same source of the second antibody to achieve the best sealing effect and avoid non-specific binding.
Abbkine IF series reagents can help you get rid of the IF experiment trouble, combined with the use of enhanced anti-fluorescence quencher to make your experimental fluorescence more dazzling!