I was having edge effect issues with DAPI staining of spleen frozen sections. After reading over some of the posts on this topic I decided to switch from Fluouromount-G w/DAPI to adding it in secondary antibody incubation. As a trial, I fixed, blocked and incubated with DAPI @ 1ug/ml for 1 hour as I would during a secondary antibody incubation. Then I did two 10' washes in PBST.
The staining looked great.
Repeated this with primary incubation o/n, then secondary with same concentration of DAPI; however did 8 10' washes to eliminate background issues associated with secondary antibody.
DAPI staining was so low, could not even focus.
Any thoughts on whether this extra washing would affect DAPI staining and if yes how best to proceed.
As another note, in my trial, I also tried staining for 5 minutes in 1ug/ml DAPI/PBS after 1 hour blocking, followed by 2 10 minute washes in PBST. Staining was not as good.
Dear Rita,
Switching to Fluoromount w/o DAPI and then adding it separately is a good decision. However, I am not sure why you are including DAPI incubation so early in your protocol and then wash so extensively? Also, why are you using PBST instead of PBS for all the washes? As Tween can permeabilize not only the cell membrane, but also nuclear membrane it is likely that this extensive washing in PBST will wash out your DAPI. If you would wash with PBS this would not happen much.
The following should work like a charm: include DAPI incubation as one of the last steps of your protocol after secondary ab incubation is done and you have done the washes after the secondary ab.
There are two options what to do:
1. Add DAPI to the last PBS(!) wash, just before mounting. Then mount.
2. (I would recommend) lets say you do 3 washes after the secondary antibody. After the 2nd wash incubate the sections with DAPI for 15 min. Then do the 3rd wash and then mount the sections. Meaning: to introduce the DAPI incubation step just before the last wash in your protocol.
Also, a few minor points that might be useful:
why so many washes after everything? How thick are your sections? Consider using less washes, but longer, so instead 8 x10' I would rather go for 3 x 15', 3 x 20' or 3 x 30' depending on how thick the sections are. I would also use PBS instead of PBST is possible, at least for the last washes. I usually included PBST (or TBST) at the beginning of the protocol to permeabilize the cell membrane in one of the steps, but then used PBS/TBS for washing.
DAPI concentration that you are using is high. You can easily dilute it at least 10x.
Good luck!
@Joanna, thanks very much for this! I actually started a primary incubation today and will implement some of your suggestions tomorrow. We were having some nonspecific binding with our secondary which is why we are washing so extensively. But I will switch to PBS for the last couple of washes before adding the DAPI.
Also as per Thermo's instructions, I resuspended DAPI powder to 5 mg/ml in DMF stored at 4C and diluted down from this stock. I've kept this stock in fridge for about a week and am assuming it should be fine? Thanks again for the feedback.
DAPI
Dear Rita,
Fingers crossed! Hope it works!
I see. If you are having trouble with high background (non-specific biding), I would suggest one or combination of the following:
1. Decreasing the tempature during II ab incubation (example: if you did 2h in RT, try ON in 4 degrees)
2. Changing the antibody. For instance Cy3 gives more background than Alexa Fluor antibodies.
3. As you did, longer washing or an additional wash is fine, but for me 8x just sounds like a bit too much. Just keep in mind to wash with PBS.
DAPI storage in 4 degrees should be completely fine. Even for a few weeks.
Good luck!
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