Proteomic analysis of PAXgene-fixed tissues.
Formalin fixation and paraffin embedding is the standard technique for preserving biological material for both storage and histological analysis. Although recent progress has been made in the molecular analysis of formalin-fixed, paraffin-embedded (FFPE) tissues, proteomic applications are a special challenge due to the cross-linking property of formalin. Here we present the results of a new formalin-free tissue fixative, PAXgene, and demonstrate successful extraction of nondegraded and immunoreactive protein for subsequent standard protein assays, such as Western blot analysis and reverse-phase protein arrays. High amounts of protein can be obtained from PAXgene-fixed, paraffin-embedded (PFPE) mouse liver and human spleen, breast, duodenum, and stomach tissues, similar to frozen material. By Western blot analysis, we found that the detection of membrane, cytoplasmic, nuclear, and phosphorylated protein from PAXgene-fixed human tissue samples was comparable to cryopreserved samples. Furthermore, the distribution of protein in PAXgene-fixed human tissue specimens is adequate for matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry for in situ proteomic analysis. Taken together, we demonstrate here that PAXgene has great potential to serve as a novel multimodal fixative for modern pathology, enabling extensive protein biomarker studies on clinical tissue samples.
Ergin
Bilge
B
Institute of Pathology, Technische Universität München, Munich, Germany.
Meding
Stephan
S
Langer
Rupert
R
Kap
Marcel
M
Viertler
Christian
C
Schott
Christina
C
Ferch
Uta
U
Riegman
Peter
P
Zatloukal
Kurt
K
Walch
Axel
A
Becker
Karl-Friedrich
KF
D000818:Animals; D015415:Biomarkers / Q000032:analysis; D015153:Blotting, Western; D015180:Electrophoresis, Gel, Two-Dimensional; D005404:Fixatives; D005557:Formaldehyde; D006801:Humans; D007668:Kidney / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D051761:Paired Box Transcription Factors / Q000032:analysis / Q000235:genetics; D016612:Paraffin Embedding; D020543:Proteome / Q000032:analysis* / Q000235:genetics; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013154:Spleen / Q000378:metabolism; D046888:Tissue Array Analysis / Q000379:methods*; D016707:Tissue Fixation / Q000379:methods*
D015415:Biomarkers; D005404:Fixatives; D051761:Paired Box Transcription Factors; D020543:Proteome; D005557:Formaldehyde