Gelatin in situ zymography on fixed, paraffin-embedded tissue: zinc and ethanol fixation preserve enzyme activity.
In situ zymography is a method for the detection and localization of enzymatic activity in tissue sections. This method is used with frozen sections because routine fixation of tissue in neutral-buffered formalin inhibits enzyme activity. However, frozen sections present with poor tissue morphology, making precise localization of enzymatic activity difficult to determine. Ethanol- and zinc-buffered fixative (ZBF) are known to preserve both morphological and functional properties of the tissue well, but it has not previously been shown that these fixatives preserve enzyme activity. In the present study, we show that in situ zymography can be performed on ethanol- and ZBF-fixed paraffin-embedded tissue. Compared with snap-frozen tissue, ethanol- and ZBF-fixed tissue showed stronger signals and superior morphology, allowing for a much more precise detection of gelatinolytic activity. Gelatinolytic enzymes could also be extracted from both ethanol- and ZBF-fixed tissue. The yield, as analyzed by SDS-PAGE gelatin zymography and Western blotting, was influenced by the composition of the extraction buffer, but was generally lower than that obtained from unfixed tissue.
Hadler-Olsen
Elin
E
Institute of Medical Biology, Faculty of Medicine, University of Tromsø, 9037 Tromsø, Norway.
Kanapathippillai
Premasany
P
Berg
Eli
E
Svineng
Gunbjørg
G
Winberg
Jan-Olof
JO
Uhlin-Hansen
Lars
L
D000818:Animals; D004396:Coloring Agents; D004792:Enzyme Precursors / Q000032:analysis* / Q000235:genetics; D000431:Ethanol; D005780:Gelatin; D007150:Immunohistochemistry / Q000379:methods; D007668:Kidney / Q000166:cytology; D008099:Liver / Q000166:cytology; D013058:Mass Spectrometry; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009206:Myocardium / Q000737:chemistry; D020411:Oligonucleotide Array Sequence Analysis; D009926:Organ Preservation / Q000379:methods*; D016612:Paraffin Embedding; D010447:Peptide Hydrolases / Q000032:analysis* / Q000235:genetics; D016707:Tissue Fixation / Q000379:methods*; D014059:Tongue / Q000166:cytology; D015032:Zinc
D004396:Coloring Agents; D004792:Enzyme Precursors; D000431:Ethanol; D005780:Gelatin; D010447:Peptide Hydrolases; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9; D015032:Zinc