IF project | Solve the difficult problems of IF one by one

Immunofluorescence (IF) is based on the principle of antigen-antibody reaction. Firstly, a known antigen or antibody is labeled with fluorescein to make a fluorescent antibody, and then the fluorescent antibody (or antigen) is used as a probe to detect tissue or cells.

In the daily IF experiment, we spent all our energy and effort to get the samples, operated with full confidence and expected the final results, but sometimes we couldn’t see any fluorescence signal or had ultra-high background signal, which made our hearts cool all at once. Even if you forget to eat and sleep, optimize the conditions, repeat the experiment, make a headache, still can’t find the cause of the problem, began to fall into self-doubt, feeling that life is not worth it!

Don’t panic, this period we will lead you to break these difficult problems one by one, to welcome the spring of IF experiment.

Difficult problem 1

Possible cause of weak or no signal

1. Improper sample preservation may lead to signal attenuation and fluorescence quenching if the fluorescent group is exposed to light for a long time.

Suggestion: Fluorescent antibody incubation and sample storage should be kept away from light. The sample can be sealed with an anti-fluorescence quench agent. Imaging and photography should be taken as soon as possible after sealing to obtain the best results.

2. Cell/tissue samples are not fresh

Suggestion: Use fresh samples to prepare slide to avoid loss of antigenicity.

3. Inadequate fixation

Suggestion: Use fixative to clean the sample quickly/thoroughly. For phosphorylation-specific antibodies, the endogenous phosphatase is inhibited with a concentration of at least 4% formaldehyde to adequately immobilize.

4. Improper use of antibodies, low concentration of antibodies or short incubation time

Suggestion: Incubation with the recommended antibody concentration in the instruction manual. The best results can be obtained when the antibody is incubated at 4°C overnight.

5. The target protein was not successfully induced to express

Suggestion: WB or other methods were used to confirm successful protein expression before IF detection, and positive control was set. Optimal treatment conditions and controls should be determined for each target.

6. Low expression of target protein

Suggestion: Consider amplifying the signal or pairing it with a brighter fluorophore.

7. Detect the absence of the target protein in the tissue

Suggestion: Add positive and negative controls.

8. Improper method of antigen repair and permeability

Suggestion: For paraffin section samples, antigen repair is required to release the masked antigen determinant. If the target protein is in the cell, the cell should be thoroughly permeated so that the antibody can fully enter the cell to recognize the target protein. Note: The sample fixed with acetone does not require a permeable treatment.

9. Incorrect use of secondary antibodies

Suggestion: Check whether the secondary antibody matches the host of the primary antibody, and use the recommended concentration.

10. Incorrect excitation wavelength

Suggestion: Ensure that the excitation light, emission light and emission filter used in the experiment match the excitation wavelength of the corresponding fluorophores. Choose the right laser to use.

11. Weak signal in multiple IHC

Suggestion: Extend section dewaxing time and use newly prepared xylene. Select the best signal amplification method.

Difficult problem 2

Possible cause of high background

1. 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.

2. 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.

3. 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.

4. Sample dries

Suggestion: Keep the sample in liquid at all times throughout the dyeing process to avoid drying out the sample.

5. Not cleaning enough

Suggestion: Full cleaning to remove residual fixatives, antibodies and other non-specific binding effects.

6. Cross-reactivity of secondary antibodies

Suggestion: Use homologous control antibodies to determine whether the secondary antibody is cross-reactive.

7. 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!

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