By Stewart Newlove, PhD and Ryan Hamnett, PhD
Immunocytochemistry/ Immunofluorescence (ICC-IF) involves using antibodies to selectively label proteins of interest in cells and visualize them with a fluorescent microscope. Below is our troubleshooting guide to help solve any issues that might be encountered with an ICC-IF experiment.
| Potential Issue | Possible Solution |
|---|---|
| Cells have detached from the coverslip | Be more gentle during washes. Reduce number of washes. Poor seeding initially, seed more cells. Cell death, check treatment/culture conditions. Coat coverslips for increased adherence. If relevant, reduce pre-extraction time or reduce detergent content of pre-extraction buffer. |
| Cells were poorly permeabilized | Increase incubation time for permeabilization. Increase detergent content of permeabilization buffer. |
| Cells dried out | Ensure that cells are covered in buffer for duration of experiment. Perform all steps in humidified chamber. |
| Cells are overfixed | Reduce incubation time with fixative |
| Too little primary antibody | Increase the primary antibody concentration and/or perform an antibody titration experiment to determine the optimal antibody dilution. Increase time of primary incubation. |
| Light exposure | Ensure that samples are kept in dark once incubated with fluorescent-conjugated secondary (or primary). Use minimum exposure times during imaging to limit photobleaching. |
| Wrong primary antibody | Confirm the antibody has been validated for ICC-IF and that antibody detects the antigen in its native form. Test other fixatives in case of epitope masking by chosen fixative. Test the antibody in a Western blot to ensure specificity and function. |
| Wrong secondary antibody | Ensure the secondary antibody is specific for the species the primary was raised in. Ensure that the secondary is working and compatible with your microscope’s filter sets by using a positive control primary. |
| Antibody storage issues | Freeze/thaw cycles may have caused the antibody to degrade. Improper dilution and handling may have caused antibody aggregation. Typically, antibodies should be aliquoted in small volumes (e.g. 10 μl) and stored at –20°C or –80°C. Avoid repeated freeze-thaw cycles. |
| Protein not present in target cells | Check of protein is expressed in experimental cell line using western blotting, or existing protein and RNA databases. Consider using positive control where abundant expression is known. Ensure the antibody is compatible with the species of the tissue sample. |
| Protein present in very low amounts | Increase sensitivity and amplification through increased secondary incubation time. Consider extra amplification using ABC, LSAB or TSA systems. |
| Epitope obscured by fixation | Try a different fixative. Perform an antigen retrieval step.* |
| Too short exposure | Increase exposure time and/or gain when imaging. |
| Coverslip wrong way up | Ensure coverslips are correctly orientated, with the cells between the coverslip and the slide. This can be checked by gently scratching the surface of coverslips under a light microscope. |
| Over-blocking | Reduce bocking time. |
* Antigen retrieval Epitopes susceptible to being masked following aldehyde-based fixation can be retrieved with incubation in an antigen retrieval buffer (100 mM, 5% (w/v) urea, pH 9.5). The buffer must be pre-heated to 95°C before coverslips are added and incubated at 95°C for 10 minutes. After, coverslips should be washed three times in PBS. | |
| Potential Issue | Possible Solution |
|---|---|
| Non-specific binding | Check background levels using absorption, isotype and secondary-only controls. |
| Poor blocking | Try increasing blocking agent concentration or changing the blocking solution. Try increasing the blocking incubation time, usually up to 1 hour. Ensure using normal serum from the same species as the host of the secondary antibody. |
| Cross-reactivity when multiplexing | Ensure that primary antibodies are from distinct host species. Ensure secondary antibodies will not recognize other secondary antibodies, e.g. donkey anti-goat will recognize goat primary and secondary antibodies. |
| Cells dried out | Ensure that cells are covered in buffer for duration of experiment. Perform all steps in humidified chamber. |
| Too high antibody concentration | Perform a dilution series to find optimal dilution for primary antibody. Dilute secondary antibody further. |
| Conditions of antibody incubations were suboptimal | Decrease the incubation time, particularly for room temperature incubations. Perform the incubation at 4°C. |
| Reactive aldehyde groups | Consider introducing 1% NaBH4 in PBS incubation step.** |
| Fixative still present | Increase washing after fixing. |
| Spectral overlap | Check the excitation and emission spectra of secondary antibodies using an online spectrum viewer. |
| Insufficient washing | Increase number of washes. Include gentle agitation during washes (rotating / rocking platform ~ 60 per minute. Increase the concentration of Tween in the PBS-T. |
| Reactivity with endogenous IgGs | Try using antibodies raised in species other than the source of the cells (especially in primary cell culture). |
** Quenching autofluorescence Aldehyde fixative-induced autofluorescence can be blocked by reducing the aldehyde group to a hydroxyl group, or by treating fixed cells with sodium borohydride. Quenching treatment time should be optimized in order to minimize detrimental effects on specific immunostaining. | |