Actually there may be several reasons. But one of the simplest ways of dealing with this problem: When you have finished mounting your section  ( be sure to flatten the sections with  wet lens paper to be sure the edges are flat (You do not need great pressure. Just enough to flatten. If the edges are not flat and they are over lapping you will have to use an ETOH gelatin solution for mounting them to the slide. This solution will shrink the sections a little so you have no over lap and the edges will stay better).  Then when the sections are dry, place the slides in formalin for  about ~8 minutes. This will fix the gelatin and make it less soluble. Rinse in DH2O. Then you can dry and keep at room temp. 

note: If you freeze the slides before they are completely dry (as you tried), the gelatin will not be completely adherent and you will have to let it dry when you take them out of the frig. (as you state), but it may not work as well. But why put them in the -20C at this point? If you are only doing cresyl violet, it is not necessary.

Hope this helps

PS:

I almost forgot. If your brains are in the embedded in M-1 matrix. And it surrounds the brain such that it is carry over to your slide. It will weaken the bond. So try to keep that off the slide when putting the section on.

thank you for your comments Frank. Actually I always let the slides try for about 45 min after mounting them and thereafter put them to the freezer.  Of course there is still some M1 matrix on them which will be washed away by xylene and ethanol washes prior to crystal violet. I use this staining just as a control and complement the FluoroJade staining which I plan to do, however, if my slices still peel off, no sense to proceed with another staining until the problem is solved. 

So your suggestion is to rinse the slides after the slices are mounted and dried, in formalin and then rinse them with H20 and let them dry and them store in the fridge/freezer? Once the brains have been perfused with paraformaldehyde and embedded in the M1 and cut and mounted, do you think additional treatment with formalin will not affect it? Thanks again for your input.

Hi Marcela,

my tip is to dry the slices overnight (or even over the weekend) at 37C and to avoid freezing them before you do the Nissl. We are always using superfrost plus glass slides, plus 0.2% Gelartin in Tris-HCl for mounting. Good luck!

Natalia

Thanks Natalia. How do you store the slides then for longer term storage?

Hi Marcela, 

usually we do the Nissl within a couple of days after mounting the slices. We use frozen free-floating sections, which we freeze at -80 after slicing and take out when we ready for mounting!

Please note Natalia's method. Free floating on to + slides is probably the best solution if you  do not have to use the gelatin.

Responding to your second question: If you just used plain gelatin, then often times researchers would  fix it  with formalin or formalin vapor. The other alternative was to use  Chrome gelatin as this was more resistant to dissolving. So with your case after your sections were allowed to dry on the slide, you would then expose the slide to the formalin.  The second exposure to formalin. Probably would not have a noticeable effect on the CV stain. But you may considering cryo-protecting your tissue before you section. As this would decrease freeze artifact.  Good luck

thank you for advice, Frank. I will try what you both suggested and see.

Is there a reason you are freezing the mounted slides? I haven't frozen slides before mounted histology (usually for Nissl). If the slices aren't adhering to the gelatin coating, that is definitely your issue (besides the freezing if it hasn't been cryoprotected, as was stated earlier). You could also try letting them sit longer at room temp, or even putting them on a slide warmer to soften the gelatin coating, which will help the sections adhere.

so, how do you use to store the slides once not all of them are going to be stained? I have generally no problem with adherence and all slides with slices look ok after rinsing in ethanols (100, 90, 70%) however, I experience problem thereafter, once I rinse in water for 1-2 min. The tissue swells and detaches from the slide. Maybe shorter rinsing in water would be appropriate but I am not 100% positive.

I only mount sections once I have a timeline for staining (and stain within 2 days of mounting), otherwise they remain in the fridge or freezer in costars in buffer solution (depending on how long you need to store them, and whether they have been cryprotected - i.e. if you sliced them on a cryostat vs. a vibratome etc.)

Hi Marcela,

Your principal problem is that your slices aren't adhering to your slides.

I don't understand why you dont wait for adhering your slices to the 2% gelatin slides longer. A couple of days at room temperature would be the minimum. Even a slide warmer would be the best solution but always for minimum 48-72h... which will help the sections adhere.

All the other considerations could help you but if you permits your slices to be well adhered rinsing in water for more than 1-2min would not be a problem.

Good luck!

Palma