The impact of tissue fixatives on morphology and antibody-based protein profiling in tissues and cells.
Pathology archives harbor large amounts of formalin-fixed, paraffin-embedded tissue samples, used mainly in clinical diagnostics but also for research purposes. Introduction of heat-induced antigen retrieval has enabled the use of tissue samples for extensive immunohistochemical analysis, despite the fact that antigen retrieval may not recover all epitopes, owing to alterations of the native protein structure induced by formalin. The aim of this study was to investigate how different fixatives influence protein recognition by immunodetection methods in tissues, cell preparations, and protein lysates, as compared with formalin. Seventy-two affinity-purified polyclonal antibodies were used to evaluate seven different fixatives. The aldehyde-based fixative Glyo-fixx proved to be excellent for preservation of proteins in tissue detected by immunohistochemistry (IHC), similar to formalin. A non-aldehyde-based fixative, NEO-FIX was superior for fixation of cultured cells, in regard to morphology, and thereby also advantageous for IHC. Large variability in the amount of protein extracted from the differently fixed tissues was observed, and the HOPE fixative provided the overall highest yield of protein. In conclusion, morphological resolution and immunoreactivity were superior in tissues fixed with aldehyde-based fixatives, whereas the use of non-aldehyde-based fixatives can be advantageous in obtaining high protein yield for Western blot analysis. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.
Paavilainen
Linda
L
Department of Genetics and Pathology, Uppsala University, Sweden.
Edvinsson
Asa
A
Asplund
Anna
A
Hober
Sophia
S
Kampf
Caroline
C
Pontén
Fredrik
F
Wester
Kenneth
K
D000906:Antibodies; D015153:Blotting, Western; D045744:Cell Line, Tumor; D005404:Fixatives; D006801:Humans; D007150:Immunohistochemistry; D009928:Organ Specificity; D011506:Proteins / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification
D000906:Antibodies; D005404:Fixatives; D011506:Proteins