I'm trying to stain mouse lung tissue IHC-IF
I fix in 4% PFA overnight immediately upon harvesting tissues
Then dehydrate/cryoprotect in sucrose gradients 15% to 30%
Then freeze in OCT media using liquid nitrogen
I'm sectioning on a cryostat at -20 degrees (5-10 um) and the tissue sections look to adhere well to the positively charged slides at this point.
Slides are kept at -20 until ready for IHC staining, but as soon as I try to remove the excess OCT with water or PBS, the tissue sections begin to wiggle loose. If I leave them in perm/blocking buffer for 30 minutes, they're half floating. And after leaving in primary antibody overnight, all tissues are floating.
How do I keep my tissues stuck to the slides? Is the PFA fixation before freezing affecting the charged slides from working like they should? Would a secondary fix step help- like a short 5 minute methanol fix?
This is very common to frozen tissues come off from the slide glasses.
First, using a good slide glass is a big help. Try using following slide glasses, they are very good. The ones with the pre-coate, adhesive slide glasses:
https://www.matsunami-usa.com/product/mas-gp/
Second, are you air drying your sections before washing off OCT with either PBS or H2O? I usually air dry my slide for 40 min to 1 hr before washing off with 1x TBST to remove OCT.
I used these methods to prevent fall-off of my cryosection, hope this can help you:
1. Air dry the sections at RT for 30min to make sure it adhere tightly to the surface.
2. You are right that the sections may fall off easily after rehydration, especially for my bone tissue. So everytime after rehydration, I will add some triton-x 100 (0.5%) onto slides for 5min, then ddH2O for 3 times X 5min. Then I bake the slides again at 60C or 37C to let them dry. This is very useful for me.
Dear Mayowa,
Baking the slices 1h @ 65 C works miracles.
Give it a try!
Cheers