Mounting

If tissue detachment occurs during 4i we recommend the following:

1. Pipette very gently. If the tissue does not show signs of detaching after the first iteration of 4i but later comes off, likely the problem is pipetting too forcefully and not with the mounting. This is the most common cause of tissue detaching.
2. Dehydrate the tissue longer. After mounting and removing the PBS the tissue can be left to dry out completely (1hour or even overnight). While this is quite effective, it can mask some epitopes and may affect the staining. This can be mitigated by “rehydrating" the tissue is PBS for at least one day after mounting and/or an initial wash with the elution buffer before staining, acting as a mild antigen retrieval.
3. Remove PBS from edged of tissue. If the complete dehydration is too aggressive, the PBS that collects at the edges of the tissue can be removed with a Kimwipe (cutting the tip of a p200 pipette tip and thread the kimwipe through to form a pointed tip provides better control). 
4. Increase incubation time for PDL coating - try up to 1 hour. It will increase the positive charge of the glass in the plate and increase the tissue adherence. However, it will make mounting more difficult so this is not recommended for delicate or thin tissues.
5. Use less Triton-X. Long incubations with high concentrations can cause epitope loss and tissue falling off