Tissue engineering: selecting the optimal fixative for immunohistochemistry.
BACKGROUND
In the immunohistochemical analysis of tissue-engineered structures, aggressive treatments for fixation and antigen retrieval can impair the quality of specimen staining and visualization.


OBJECTIVE
We hypothesized that the adequate choice of fixative and antigen-retrieval method might improve the quality of immunohistochemical staining.


METHODS
Tissue-engineered vascular grafts were fixed using formalin, Carnoy's, or HOPE(®) fixative. Antigen retrieval was performed where necessary and samples from each group were stained using hematoxylin and eosin to assess overall tissue preservation. For a set of proteins relevant to cardiovascular tissue development, immunohistochemical staining was applied to formalin-, Carnoy's-, and HOPE-fixed specimens to allow a comparative analysis.


RESULTS
In tissue-engineered constructs, antigen retrieval methods necessary after formalin fixation led to significant destruction of the overall tissue structure. Carnoy's fixation resulted in good overall tissue preservation and adequate results for immunohistochemical staining of alpha-smooth muscle actin (α-SMA), vimentin, type I collagen, elastin, and laminin. HOPE fixative led to a loosened tissue structure and a swollen appearance but showed adequate results for staining against type III collagen and elastin. Formalin fixation without antigen retrieval led to inadequate visualization of α-SMA, vimentin, type I- and type III collagen, and laminin.


CONCLUSIONS
Based on the present study, we recommend that Carnoy's fixative is employed for the preservation of tissue-engineered constructs to allow immunohistochemical analysis of type I- and type III collagen, elastin, laminin, α-SMA, and vimentin. However, it is clear that the technique requires optimization based on the particular tissue engineering application.
Department of Tissue Engineering & Textile Implants, Applied Medical Engineering, University Hospital Aachen, Aachen, Germany.
Sabine
S
Koch
Nadine
N
Stappenbeck
Christian G
CG
Cornelissen
Thomas Cormac
TC
Flanagan
Petra
P
Mela
Jörg
J
Sachweh
Benita
B
Hermanns-Sachweh
Stefan
S
Jockenhoevel
D000818:Animals; D002478:Cells, Cultured; D007150:Immunohistochemistry; D012756:Sheep; D023822:Tissue Engineering