Hi Ahmed, you are right, the brain slices bind together, like adhesive tape. I've never heard of using coconut water, do you think it would change the results of the Immunohistochemistry? The problem is that I haven't done the procedure, because I have to extend the slices to get a good antibody impregnation.

have you tried it before? 

Thanks for taking the time to answer. 

I've seen that sheets you are talking about, but I don't think they work with free floating cuts, I think they are special for freezing slices. And also, I would have to expand the brain slices in order to put them between the paper, and some of them are very enmeshed.

Do you think that if I apply a few drops of fixative solution (paraformaldehyde 4%) in to the phosphate buffer, the slices become harder, so I could handled them easier without brake them?

This is unusual slice behavior. I would first check whether solution concentrations/pH are correct (PBS, formaldehyde (is it in PBS?), sucrose). 

Hi Victor. Yes, the solution is phosphate buffer 0.1M. I haven't seen if the pH has change, but I will. Maybe you're right and the problem is just because of that. 

A few drops of 4% PFA into the buffer will help reduce the amount of sticking to each other, and to the bottom/walls of the dish or well.  I do this for vibratome sections of CNS.  Hope this helps and good luck with your experiments.

Hi Yvette, I assume that the slices break while you try to separate them before mounting on slides, is it? As written above, maybe your PB is not quite right, maybe your slices are too thin, maybe you slice them a little too fast which makes them more fragile, maybe the blade angle on the cryostat is not as perfect as it could be....

When mounting, I use a large petri dish, dump all the slices from a well together in the center and they separate almost on their own. Make sure to keep your paint brush as parallel the slice as you can, to prevent making wholes. Hope this helps

Hi Rachel and Adam, thank you for the advices, they are very helpful (: I did put them some drops of fixative solution and they became a little less sticky. Also, I check the pH of the buffer solutions and it was ok. My thesis director says that it could be because of the blade of the cryostat, it was kind of old. But anyway I'm saving the slices that look better. Hope they survive the next steps of my protocol. 

Store them separately. We used the plastic boxes for eggs. Moreover you can store the boxes by placing the halves on each other and put themin a plastic sac. These egg boxes are present for small eggs and large ones (small slices versus large slices). The industries that produce them were always helpful. They are not expensive. If you can fixated the sections before use glueing is overcome. Succes. Enrico Marani

Hi Yvette, how to you cryoprotect your brain after fixing it? I think that by improving the quality of your slices, you may decrease the issue with them sticking.  You are welcome to email me as well.

Dear Yvette,

I second Enrico's suggestion to store the slices separately.  It is also an easy way to keep the slices in serial order.  Depending on the size of the slices, we us multi-well tissue culture plates.  Since we don't use them for culture, we just reuse them after washing them. 

You are so kind, thanks for helping me (: I too use multi-well tissue culture plates, but I don't put every slice separately. I put almost 20 slices in every well. I think I must be more careful when cutting and store them, because it hadn't happen to me before. Thanks Rachel, after fixing, I put them in a train of sucrose solution, 10%, 20%, and 30%, around 3 days in each one, is it ok, or they need more time?

Thank you Michel and Michael, I'll take your advices into account. In fact I already add some drops of fixative solution in the wells where I kept my slices, and after a few days they were more handable (:. I also knew that the PFA diminishes the affinity of the antibodies, but I applied the same cuantity in all the wells in order to 

After you do sectioning using cryostat and as you said you store the sections in phosphate buffer. Here i would like to add that, add more volume of buffer per well (eg 2-3ml in each well of 6 well plate). More volume provides more space for sections when you store. Next thing, after sectioning it is always better to mount the sections on slide and store in -80. 

Hope this helps.

Hi Yvette,

I am also running into a similar issue with my cryosections. Did you notice any difference in your staining when you added a few drops of PFA vs not?