Terapia prechirurgica della fibromatosi uterina - FedOA - Università ...

Italy). For that concerning the vascular evaluation, immunostaining for CD34, as a pan-endothelial
marker, was performed with an anti-CD34 primary antibody (QBEnd 10 monoclonal mouse, 1:200;
Dako, Carpinteria, CA).
Incubation was carried out overnight at 4°C, in a moist chamber. After incubation and washing,
sections were incubated with biotinylated link antibodies and peroxidase-labeled streptavidine
(LSAB-HRP, Dako, Italy). 3,3 Diaminobenzidine (DAB, Vector Laboratories, Burlingame, CA) with
0.3% H2O2 was used as substrate chromagen solution. After nuclear counterstaining with
hematoxylin, sections were mounted with synthetic medium (Entellan, Merck, Dermstad, Germany).
PCNA was observed as nuclear staining in leiomyomas smooth muscle cells. Only cells exhibiting a
definite staining were judged as positive. PCNA expression was semi-quantitatively evaluated using
the following score: + (< 5% of stained cells), ++ (5-10% of stained cells), +++ (>10% of stained
cells).
CD34 was observed as cytoplasmic staining in blood vessels endothelial cells. CD34 expression was
simply scored as negative or positive. The number of CD34 positive blood vessels, also including
mono-endothelial ones, was determined in at least five different fields of representative areas, at 100x
magnification (0.25 x 0.35 mm).
Sections were examined and immunostaining was graded without previous knowledge of the clinical
data of the patients.
Statistical analysis was performed using the Student t test. Levels of statistical significance were set at
p-values