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.
Frota
Ana Carolina de Arantes
AC
Federal University of São Paulo, UNIFESP, São Paulo, Brazil. carolafrota@gmail. com
Lima Filho
Acácio Alves de Souza
AA
Dias
Ana Beatriz Toledo
AB
Lourenço
Andréia Cristina Dos Santos
AC
Antecka
Emilia
E
Burnier
Miguel N
MN
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