After immunohistochemistry of previously fixed in PFA and EtOH and then frozen 20 μm sections of zebrafish brain, DAPI staining is very weak (right) compared to the same sections stained without preceding IHC (left). Please also take into account that the exposure time for the right image is ten times longer than for the left one – so in reality, it is much dimmer than in the picture. The concentration of stock DAPI (Invitrogen™ D3571) dilactate solution is 5 mg/mL DAPI (10.9 mM), and the final working solution is 300 nM (i.e., 1:36,333.33 from the stock solution, as per the manufacturer's recommendations). Incubation time is 5 minutes, followed by 3 washes in PBS for 1 minute each and mounting with VECTASHIELD® Vibrance™ Antifade Mounting Medium. Both stock solution aliquots and the working solution are freshly prepared. No antigen retrieval was used for this IHC, and for blocking, 0.1% Triton + 5% NGS in PBS was used. Please recommend if you have any ideas on how to improve staining. Thank you!
Probably the emission of DAPI covers excitation of dye you have used. Which dye are you using? You can check spectra using this tool: https://www.thermofisher.com/order/fluorescence-spectraviewer/#!/
Hello Piotr, thanks a lot for your reply! I'm using a pretty standard combination of Alexa 594 (red) and Alexa 488 (green), so theoretically it shouldn't be a problem.
DAPI is an AT intercalating dye so depends on the presence of double stranded DNA to give a signal. It could be that the DNA is being denatured by solvent, high or low pH, detergent, or high salt experienced during your IHC. If you are using serum as a blocking reagent there may also be nucleases present. Exposing a DAPI stained sample to high light levels during wide field microscopy can also bleach the dye and/or shift its emission wavelength further into the green region of its emission spectrum.
Did you have a negative sample without antibody, only DAPI stained during your procedure? As you have shown it should work. I remember that some years ago I had a similar problem with DAPI + Alexa Fluor 488 or FITC (I‘m not sure, it was in previous lab). That time I switched to Hoechst 33342 and I had much better signal. I am not sure why, but it might be what’s David G Spiller wrote. If you have possibility you might try. You can also try staining without Alexa Fluor 488, only with Alexa Fluor 594, it will show whether it’s antibody dye or procedure fault.
Piotr Lewandowski and David G Spiller, thank you so much for all your insightful advice! I will definitely perform all these control experiments: making a negative control with blocking solution only, staining with Alexa 594 only, replacing the normal goat serum I’m using for blocking solution with an alternative, and not using Triton X-100 as a detergent. I can exclude the possibility of photobleaching since I captured these images within one hour to one day after staining, without any prior light exposure, including during the staining. Also, I use PBS with freshly adjusted pH, so that’s likely not an issue. No additional salts or temperatures higher than room temperature were used (though I performed antigen retrieval at 70°C, but the results were as poor as without it). It seems to me that my samples might be at double risk due to the unusual protocol of their fixation, and now something very minor that usually doesn’t affect the staining is affecting mine. This fixation protocol included 24 hours of 4% PFA fixation, followed by storage in liquid 75% EtOH for more than one year. So, they are overfixed, but the EtOH likely already provided sufficient permeabilization for good staining since it dissolves lipids. Therefore, it might not make sense to add Triton X-100 to my blocking solution at all. I’ll share updates once I finish the additional experiments!
Summing up my recent batch of experiments:
1. Staining with Alexa 594 does not affect DAPI staining differently from Alexa 488; the result is equally poor.
2. Alternatives to normal goat serum yield the same poor result.
3. Omitting antibodies from the reaction, i.e., performing just a ‘sham’ IHC before DAPI staining—with all incubations and washes but with a blocking solution only — still compromises DAPI staining. It is hard to determine why, whether it is due to quenching the signal, denaturing DNA, or just due to high background (which might not be the case since the overall signal is too weak — exposure time is 600 ms).
4. Omitting Triton X-100 as a detergent does not change anything.
5. Finally, what made the staining perfect even after this ‘sham’ IHC (see the attached photo) is a profound increase in DAPI concentration (from 1:36K to 1:1K) and duration (from 5 min to 15 min). It is hard to determine which factor—duration or concentration—was more critical for successful staining, so I plan to test incubation for 15 minutes at 3K, 10K, and 15K, and possibly try lower concentrations for 30 minutes. It is also time to add my target antibodies to see if they interfere with DAPI staining as well. I will try to update you on my results.
Update:
Dissolving the stock solution in PBS takes about 5 minutes with very vigorous shaking. It's better to use a transparent flask to ensure that the solution is fully dissolved (i.e., no yellow DAPI particles remain). However, be sure to keep it in darkness during the process. Our previous issues with insufficient staining were likely due to incomplete dissolution. When properly controlled, diluting the stock solution at least 1:10,000 and staining for 15 minutes yields good results. Adding antibodies did not interfere with the staining achieved at this dilution. Thanks again to everyone who provided feedback!
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