Hello everyone,
I am currently having issues with my brain slices edges unsticking from the Superfrost Plus slides (not expired) and folding during my immunohistochemistry experiments.
The brains were perfused with NaCl and stored at -80°C. I then cut them using a cryostat at a 16um thickness after coating them with OCT. The temperature of both the chamber and object do not vary (-20°C) and I let the slices dry off a bit so that they adhere to the slide before storing them back at the -80°C.
However when I start immunohistochemistry and fix theses slices in a PFA bath for 10 minutes, the edges of the slices detach from the slide (without the slice actually falling off), and the edges can fold onto the rest of the slice, preventing good image analysis. Furthermore the edges (the cortex mostly) also become damaged (see picture) due to the fact that they do no stick to the slide while I change solutions in my experiment.
Any guesses as to what could cause this and how it could be improved?
Thanks !
Hello Thais Lestra,
one reason for your prombles could be that the sections were not transparent after you have transfered them on the slide because the time was to short. If this is so you get lost the sections during the following process steps. Also helpful is that you equlibrate your sections to room temperature (it will take 45 min upto 1 hour) before you start with your immuno or Insitu. What to do? After you have transfered the sections on prcooled slides thaw them while you are wiping with your finger tip the backside of the slide until they becom transparent. Put the slide into a box which you have placed in the crtyostate.
The second reason could be the quality of your sections . Check whether the border of your brain sections have this lace like appearence before you start with your immuno. You can see it when you hold the slide against light or check it under the microscope. If this is the case you have to improve your freezing protocol of your brains because you have a so called cryoartefact. Put the brains in -80°C cold isopenthan before storing them into the -80°C freezer because a short freezing time is importent for shock freezing. Good luck!
Hi Thaïs Lestra
* Please check if the same is occurring during cryotome sectioning or is it occurring during the staining steps?
* One of the primary reason could be inadequate drying of the slides post sectioning. Dry the slides at 37o C for an hour and store the same in -80.
* If the folding is occurring post fixation with PFA, I would suggest you to try ice cold methanol fixation for 10mins, instead of PFA.
Hope it helps.
Thanks,
Hello Thaïs Lestra,
The most likely reason is the perfusion for the brain, which has a high content of lipids. We have started to use hydrophilic plus slides such as Leica Apex or TOMO slides (Adhesion slides TOM-11) for brain tissue. Perfusion, PFA in PBS or any other water based solution is reducing the capacity of the brain tissue to stick. It is also beneficial to use +37C incubator before equlibration to RT staining.