AGs, spermatic ducts (SDs), and ejaculatory bulbs (EBs), were
dissected out and fixed overnight in Bouin’s fixative, at 4 ◦C. They
were then washed with 70% ethanol and dehydrated through a
series of ethanol, transferred into toluene, infiltrated with melted
paraffin, and embedded in paraffin blocks. Serial sections of 5m
thick weremadeat the mid-region of the AGs, using a LeicaRM2235
microtome, and then placed on silane coated-slides and air dried
overnight. The paraffin sections were deparaffinized with xylene,
then rehydrated through a series of ethyl alcohol, stained with Harris’s
hematoxylin for 1min and counterstained with eosin for 2min.
They were then dehydrated with a series of ethyl alcohol, cleared
with xylene, and mounted in Permount (Bio-Optica, Milan, Italy).
Sections were examined using a Nikon eclipse E600 microscope
and photographed with a Nikon digital camera DXM1200.
The numbers of cell types present in the AGs were
quantified from uniform cross-sections, using ImageJ software
(http://rsbweb.nih.gov/ij/index.html), then recorded as percentages
of the total cells. In the BrdU assay groups (see Section 2.5),
the numbers of dividing cells per mm2 area were counted by the
same method.
AGs, spermatic ducts (SDs), and ejaculatory bulbs (EBs), weredissected out and fixed overnight in Bouin’s fixative, at 4 ◦C. Theywere then washed with 70% ethanol and dehydrated through aseries of ethanol, transferred into toluene, infiltrated with meltedparaffin, and embedded in paraffin blocks. Serial sections of 5mthick weremadeat the mid-region of the AGs, using a LeicaRM2235microtome, and then placed on silane coated-slides and air driedovernight. The paraffin sections were deparaffinized with xylene,then rehydrated through a series of ethyl alcohol, stained with Harris’shematoxylin for 1min and counterstained with eosin for 2min.They were then dehydrated with a series of ethyl alcohol, clearedwith xylene, and mounted in Permount (Bio-Optica, Milan, Italy).Sections were examined using a Nikon eclipse E600 microscopeand photographed with a Nikon digital camera DXM1200.The numbers of cell types present in the AGs werequantified from uniform cross-sections, using ImageJ software(http://rsbweb.nih.gov/ij/index.html), then recorded as percentagesof the total cells. In the BrdU assay groups (see Section 2.5),the numbers of dividing cells per mm2 area were counted by thesame method.
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