HOPE technique enables Western blot analysis from paraffin-embedded tissues.
In contrast to the spectrum of biochemical analyses of fresh material, that of archived specimens is widely restricted. Fixation of specimens with formalin, the most commonly used fixative, usually prevents further molecular analysis, since it leads to degradation of nucleic acids and denaturation of the antigenic determinants of proteins. To overcome these problems, the Hepes-glutamic acid buffer mediated Organic solvent Protection Effect (HOPE)-fixation technique has been developed, which preserves nucleic acids and antigenic determinants of proteins, thus expanding the applicability of immunohistochemical methods. In this study, we investigated whether HOPE-fixed tissue can be analyzed by Western blotting. Furthermore, a comparison with conventionally fixed and frozen material was made. The specimens used were tumor-free and obtained from lobectomies for lung cancer. All four antibodies tested, i.e., antibodies specific for focal adhesion kinase, surfactant protein A, PI-3-kinase, and IKKalpha, worked well if used for immunoblotting of HOPE-fixed and frozen tissue. By contrast, these antibodies showed no or only very weak specific binding if formalin-fixed specimens were analyzed. Our findings show that HOPE fixation maintains the antigenicity of proteins better than formalin fixation. The possibility for performing Western blotting with archived paraffin-embedded specimens extends the options for diagnostic and scientific analyses of fixed tissues.
Uhlig
U
U
Division of Pulmonary Pharmacology, Research Center Borstel, Germany.
Uhlig
S
S
Branscheid
D
D
Zabel
P
P
Vollmer
E
E
Goldmann
T
T
D015153:Blotting, Western / Q000379:methods*; D015925:Cryopreservation; D005404:Fixatives; D006801:Humans; D008168:Lung / Q000737:chemistry / Q000378:metabolism; D016612:Paraffin Embedding; D011506:Proteins / Q000032:analysis / Q000378:metabolism; D012680:Sensitivity and Specificity; D013997:Time Factors; D016707:Tissue Fixation / Q000379:methods*
D005404:Fixatives; D011506:Proteins