The AMeX method: a multipurpose tissue-processing and paraffin-embedding method. Extraction of protein and application to immunoblotting.
The authors have previously reported a new fixation and paraffin-embedding method (the AMeX method), which preserves many antigens as well as high molecular-weight DNA and RNA that are normally destroyed by the routine formalin fixation and paraffin-embedding process. In the present study, the authors analyzed the preservation of protein suitable for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting in tissue fixed by the AMeX method. The method used for extraction of protein from AMeX-processed tissue sections after deparaffinization was the same as that for extraction from fresh tissues. The total amount of protein extracted from 50-mg (wet weight) AMeX-processed mouse liver tissue was the same as that from fresh tissue. The electrophoretic mobility and staining intensity of protein on SDS-polyacrylamide gel, and the immunoblotting pattern and staining intensity with several antibodies, were identical for both AMeX-processed and fresh tissue. Degradation of protein was minimal for storage periods of 2 years in paraffin block. The authors also showed that pellets of cultured cells can be processed by this method for immunologic analysis. This new fixation and paraffin-embedding method is a useful tool for obtaining information on correlations between morphologic features and immunochemical and molecular biological data.
Sato
Y
Y
Pathology Division, National Cancer Center Research Institute, Tokyo, Japan.
Mukai
K
K
Furuya
S
S
Kameya
T
T
Hirohashi
S
S
D000818:Animals; D015925:Cryopreservation; D004591:Electrophoresis, Polyacrylamide Gel; D006652:Histological Techniques; D015151:Immunoblotting; D007158:Immunologic Techniques; D051379:Mice; D008807:Mice, Inbred BALB C; D016612:Paraffin Embedding / Q000379:methods*; D011506:Proteins / Q000032:analysis*; D013194:Staining and Labeling
D011506:Proteins