Freeze-drying as an alternative method of human sclera preservation.
OBJECTIVE
To compare the effect of preserving sclera samples in either 95% ethanol or freeze-dried.


METHODS
Ninety-six samples of human sclera were studied. Half of them were freeze-dried and half preserved in 95% ethanol. Preservation periods of 18, 45, 90 or 174 days were studied. Automated immunostaining was carried out in the Ventana BenchMarkR LT platform using collagen 1 and fibronectin antibodies. Histological staining was also performed with hematoxilin-eosin and Masson trichrome. Samples were classified according to the degree of collagen fiber parallelism (0-2), intensity of Masson staining (0-2), and the expression of both antibodies (0-3). Friedman and Wilcoxon tests were applied to compare preservation methods and p-values below 0.05 were considered to ensure statistical significance.


RESULTS
Relevant results were found in three situations: (i) Friedman's test showed better collagen fiber integrity in the freeze-dried group rehydrated after 174-days as compared to the 90-day group; (ii) Wilcoxon's test showed better collagen fiber integrity in the freeze-dried group after 18 and 174 days as compared to the ethanol group; (iii) the freeze-dried group disclosed higher immunohistochemical expression for COL-1 antibody in the sclera samples rehydrated after 45, 90 and 174 days as compared to the ones rehydrated after 18 days.


CONCLUSIONS
Histological and immunohistochemical analysis showed freeze-drying to be a superior method for sclera preservation as compared to 95% ethanol. This technique provides an easy method to manipulate tissue, with longer shelf life, and storage at room temperature.
Federal University of São Paulo, UNIFESP, São Paulo, Brazil. carolafrota@gmail. com
Ana Carolina de Arantes
AC
Frota
Acácio Alves de Souza
AA
Lima Filho
Ana Beatriz Toledo
AB
Dias
Andréia Cristina Dos Santos
AC
Lourenço
Emilia
E
Antecka
Miguel N
MN
Burnier
D000891:Anti-Infective Agents, Local / Q000737:chemistry*; D000906:Antibodies / Q000276:immunology; D024042:Collagen Type I / Q000276:immunology / Q000378:metabolism; D000431:Ethanol / Q000737:chemistry*; D005353:Fibronectins / Q000276:immunology; D005612:Freeze Drying / Q000379:methods / Q000592:standards; D006801:Humans; D007150:Immunohistochemistry; D011446:Prospective Studies; D012590:Sclera / Q000276:immunology / Q000378:metabolism; D013194:Staining and Labeling; D018709:Statistics, Nonparametric; D013997:Time Factors
D000891:Anti-Infective Agents, Local; D000906:Antibodies; D024042:Collagen Type I; D005353:Fibronectins; D000431:Ethanol