DRG staining - samples fall of slides


#1

I’ve been staining for DRG directly mounted on to slides…the issue I’m having is that the DRGs often slide off the slide. They are on positively charged slides, so I can’t figure out why this is happening.

Does anyone have any tips? Subbed slides? Extra positively charged slides? Anything!


#2

Hi @ShanTan. I’m sorry that’s happening to you. That happens to me occasionally, but not very much.

Are you using Superfrost Plus slides? That’s the first thing. 12 um slices?
While I’m slicing I leave the slides outside the cryostat and this allows them to air dry. I’m not super rigorous about time, but it’s at least 20 minutes. Some people gelatin coat their slides, but I’ve never found that necessary.

Are you embedding in OCT?

@tberta Any thoughts?


#3

Thanks for this - I am embedding in OCT, cutting 14 um slices, and using SuperFrost slides…

When you rinse your slides with PBS, is there a particular technique you use to try and avoid losing samples?


#4

Hey @ShanTan

Superfrost or Superfrost Plus? That’s a big difference and it’s happened before that I use just Superfrost (by accident) and then have all my tissue fall off. Check to see if there are two plus marks on the side opposite the white frosted area.

Try 12 um. For PBS rinses, nothing special. I use this easy dip system (https://www.emsdiasum.com/microscopy/products/histology/staining.aspx) and just take the slide rack out, replace the PBS, and then put back in.

Have you done much DRG slices/IHC before? Do you always have this issue, or this is your first time? And what do other people in your lab do? Like I said, this happens only very rarely for me.

If you are using Superfrost Plus, maybe try a new batch?


#5

From my own personal experience, it is important to use Superfrost Plus slides (https://us.vwr.com/store/catalog/product.jsp?catalog_number=48311-703) and allow the sections to air dry for at least 10-15 minutes outside of the cryostat. We cut our sections at 12 um and rinse them in jars, but these factors are generally irrelevant if you use the aforementioned slides and air dry.


#6

Thanks @tberta. I agree.


#7

@ShanTan Did you figure out the problem?


#8

I did - Thanks so much for your help! Letting them dry for an appropriate amount of time seemed to make all the difference.


#9

Just another story about how important it is to use the Superfrost Plus:
A colleague came to me yesterday frustrated that during some gentle wash steps, all his spinal cord and brain slices fell off the slide. I asked him if he dried the tissue beforehand, and he said he did, for an hour in fact. At that point, I knew exactly what the problem was. I asked him to show me the slide, and sure enough, it was a regular Superfrost slide, uncharged (not plus). I feel like this happens to everyone at some point. It definitely happened to me before, which is why I’m really vigilant now. So lesson: always check your slides.