I've seen something similar in imaging other people's slides. My thought is that the DNA is getting smeared, either during the cutting of the sections by the knife, or maybe during mounting of the coverslip if there is some sideways movement.

Hello Austin, may be some things can help you:

1. use a normal over view staining like H&E or if it is brain tissue cresyl violett

to check the quality of your tissue. As Christopher mentioned it, it seams to be that the tissue is damaged . This could lead to such staining results you have presented.

2. Check the data sheet of your used antibodies whether they are suitabel for unfixed and/or aceton fixed tissue.

3. I think the perfusion with PBS for 5 min is too long.

To me this doesn't quite look like smearing of a stain. The edges are too sharp, the structure is too well defined. A smear rarely has this kind of structural integrity to it. You've mounted it in ProDiamond with DAPI though. What this means is that there is always DAPI around preferentially accumulating in regions it has high affinity for. It will also stain RNA, although in, for example, a cell grown on a coverslip the intensity of this fluorescence is so much lower than that from the nucleus that you never really see it.

The slides are also quite old. I know you've said that they're stored at -80C, but this is just what you've been told. Perhaps a collaborator left them out on the bench over the weekend once, they may have a -80C freezer which really should be replaced as it isn't uniformly keeping everything at that temperature. The slides have also been shipped around. There is a possibility that there is some sort of bacterial/fungal growth in those regions but at that resolution you wouldn't be able to tell.

I'd suggest doing two things. The first is zooming into a section where that streaking is quite pronounced and taking the highest resolution image you possibly can in order to see if the stain is uniform or punctate. This might hint at some sort of contamination.

The second is to ditch the ProDiamond with DAPI either get the pure chemical and make up the stock yourself or buy one that is preprepaired. If you see papers where people have used, for instance, a 1/10,000 dilution of their stock and stained for 10 minutes. Dilute the DAPI at say 1/100,000 and add it along with the primary antibody. With all the subsequent washes and incubations you will thoroughly rid the tissue of any DAPI that's not strongly bound to something. You should end up with pretty close to zero background and intensly bright staining everywhere you expect (ie the nucleus).

If the intensity of the streaks is still as bright as the nuclei under these conditions it would be hard to argue that it's binding to anything other than DNA which for whatever reason has ended up in those locations. If the difference in intensity between the nuclei and the streaks has greatly increased perhaps its due to huge amounts of RNA in those areas.

You won't get a conclusive answer with one or two slides but it may help point you in the right direction.

Hi, Austin Jones; your protocols look perfect. All suggestions Matthew BorrelliChristopher B O'Connell Howard Vindin Ute Neubacher are lovely. Furthermore, it has been observed that DAPI staining appears to be specific and satisfactory after preparing slides and conducting imaging. However, storing the slides at 4 degrees Celsius overnight can compromise the staining quality. It is evident that prolonged storage duration may adversely affect the integrity of the DAPI stain.