Histological and immunohistological that analysis The explanted matrigel plugs were fixed in buffered formalin solution (10%), dehydrated using a graded ethanol series and automatically embedded in paraffin according to standard procedures. Paraffin sections of 4 ��m thickness were cut, rehydrated and stained with hematoxylin and eosin staining according to standard procedures. To identify fibers secreted by fibroblasts the Elastica van Gieson (EvG) staining was performed according to laboratory standard methods. The immunohistological detection of CD31 and CD34 was performed with the peroxidase method by using DAB as chromogen. For this purpose monoclonal antibodies against CD31 (dilution 1:400, Santa Cruz) and CD34 (dilution 1:200, Santa Cruz) were used.
The immunohistological staining was automatically performed with help of an Autostainer plus (Dako) and the appropriate Dako-Staining kits. After the immunodetection the nuclei of cells were stained by hemalaun. Transmission electron microscopy (TEM) For transmission electron microscopical analysis samples of 0.5 �� 0.5 cm were cut from the explants, fixed in buffered glutaraldehyde (2.5%) overnight, contrasted with OsO4 and then embedded in Agar 100 (Plano), which polymerized for at least 24 h. After trimming first semi-thin sections were performed to identify the region of interest, which was then cut as ultrathin sections with the Ultracut E microtome (Leica). Transmission electron microscopical analysis was performed with a Phillips EM 410 (Phillips). Visualization took place at 80 KV and images were photodocumented.
Statistical analysis All experiments were performed at least in triplicate. Quantifications are expressed as mean �� standard error of the mean (SEM). For comparison between two groups the Student’s t-test was used. A difference between experimental groups was considered significant with a confidence interval of 95%, whenever p < 0.05. Acknowledgments The work performed at INEB was financed by FEDER funds through the Programa Operacional Factores de Competitividade��COMPETE and by Portuguese funds through FCT��Funda??o para a Ci��ncia e a Tecnologia in the framework of projects PEST-C/SAU/LA0002/2011 and POCI/SAU-BMA/55556/2004 and the PhD scholarship SFRH/BD/40421/2007 for S.G.G. Disclosure of Potential Conflicts of Interest Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed.
Footnotes Previously published online: www.landesbioscience.com/journals/biomatter/article/20063
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