In the remaining four experimental groups, the canals were emptied using PTR alone (group 2), with the addition of the solvent xylol
(Bioquimica Industria Farmaceutica, Sao Jose do Rio Preto, SP, Brazil) (group 3), orange oil (Citrol; Biodinamica Quimica and Farmaceutica, Ibipora, PR, Brazil) (group 4), and eucalyptol (ASFER Industria Quimica, Sao Caetano do Sul, SP, Brazil) (group 5).
At diafanization, to make the tissue semi-transparent, the alcohol present in the tissues was replaced by xylol
and the samples were kept in alcohol: xylol
(1:1) for one hour and then immersed in two sequences of xylol
for 30 minutes each.
and warm paraffin were then added into the tube.
Briefly, the sections were dewaxed and rehydrated in xylol
(Sigma, Germany) and a graded series of 100%, 90%, 80%, and 70% ethanol.
Formalin-fixed, and paraffin-embedded sections were deparaffinated (2 x 2 min xylol
) and rehydrated.
and alcohol series, heat-mediated antigen retrieval was performed in 10 mM sodium citrate buffer (pH 6.0).
(112) Still, other studies identify xylene and chloroform (93) or just chloroform (compared with xylol
after tissue digestion with trypsin) (113) as superior clearing agents.
After that, they were washed in running water, dehydrated in increasing concentrations of alcohol to 100%, diaphanized in xylol
and embedded in Paraplast[R].
These mice were sacrificed and the specimens from testicular tissues were fixed in 10% neutral buffer formalin dehydrated in ascending grades of ethanol alcohols cleared in xylol
casted blocked cut at 2-5 m thickness and stained with hematoxylin-eosin for microscopic examination.16Statistical analysis: All the data thus obtained was statistically analyzed by applying one way analysis of variance (ANOVA).
The trimmed tissues were first washed with tap water followed by dehydration through a graded alcohol series and then passed though xylol
and paraffin series before being embedded in paraffin.
The slides were rinsed thoroughly in distilled water, counterstained with Harris hematoxylin for 30 s, washed in distilled water, dehydrated in graded alcohols, cleared in xylol
, and were then mounted in Entellan (Merck, Germany).
Paraffin sections (4.0 [micro]m thick) were mounted on slides, deparaffinized in xylol
, and rehydrated in a descending ethanol series for immunostaining.