Gentle epitope recovery in weakly fixed or fragile tissues
-Temperature sensitivity of an epitope (heating over 100 degrees leads to irreversible denaturation of the protein without subsequent proper re-folding). This is a quite rare case, but may be observed with i.e. antibodies prepared against native molecules, such as old generation mAbs to human and mouse CD markers
- The tissue was not properly fixed, or is in general is quite fragile, with an insufficient density of components to support the structure and morphology during the heat-induced epitope recovery.
Some companies argue that the solution is to use epitope recovery units with different temperature regimes. Usually these models are more expensive, and you have to keep records for the temperature regime for every individual tissue sample.
In actuality, the solution is quite simple and may be applied in Retriever, or any other HIER system: Glycerol. Yes, ordinary glycerol.
The addition of any percent of glycerol to the buffer will effectively decrease the boiling temperature of the recovery buffer without any effect on its recovery properties.
In our lab we usually make additional versions of the buffer that contain 5 or 10% of chemically pure glycerol. Just add glycerol to required concentration and check (and required - re-adjust) the pH of the buffer.
You will receive a standard version of buffer for gentle recovery (the higher is glycerol content, the more gentle will be the recovery). In real life we have never had to go over 10% of glycerol.
P.S. A small additional remark: although the pressure in units like Retriever effectively prevents bubble formation during the heating cycle, for very gently tissues a small extra care can make a large difference. The buffers, especially when stored already in diluted form in the refrigerator, tend to absorb extra air from the atmosphere. Therefore, for a very gentle tissue samples it is best to prepare the working dilution of the buffer and to de-gas it under vacuum for 5 min, allowing the excess of dissolved air to leave the buffer