I have paraffin-embedded tissue sections that have been melted onto charged slides. I wash in xylene, and rehydrate in graded dilutions of ethanol (100% - 70%, 0%). Then I put the slides vertically in antigen retrieval solution (EDTA 1mM, pH 9.0, SDS 0.05%) at 90-95oC for 30 minutes.
However, every time I've done this the sample comes off the slide during this last step. Why does this keep happening?? Any advice would be much appreciated!
Are you microwaving the slides in antigen retrieval buffer or just heating them (say in a pressure cooker?) If you are microwaving for 30 minutes it could be a bit excessive. Sometimes excessive boiling can detach the tissue. Try using coated slides instead or reducing the microwaving time. Or you can heat up in stages of say 5- 10 minutes, rest a couple of minutes then heat again so excessive boiling doesn't occur in your solution. Occasionally if you don't allow the slides to cool sufficiently afterwards and transfer them to another buffer (say PBS at RT) the quick temperature change can also detach them.
Instead of using just charged slides such as poly-L-lysine, try something like a silane coated slide that attaches the tissue section using both charge attraction as well as covalent bonding. Good luck!
Poly-L-lysine-coated slides are the best for IHC. Precoated slides are available at Dako. Microwave can overcook your tissues.
The best is to use a pressure cooker, (ten mins with stopper and keep it for gradual cooling to room temperature), and proceed with blocking and primary ab.
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