The observer places the eye at the exit pupil of the eyepiece, the point at which the light rays leaving the eyepiece come together.
Tumour vascularity was counted by scanning at low power (× 40 100) for the three areas of highest vascularity before using a 25-point Chalkley point eyepiece graticule at × 250 (0.155 mm) over these hot spots.
Axonal densities were counted using a 24-point eyepiece from Olympus (Hamburg, Germany), and the number of points crossing axons was measured as a fraction of the total number of points on the sterological grid [ 27].
In brief, testicular cross sections were examined using a Leitz 363 plan apo objective lens (x63) fitted to a Leitz Laborlux microscope and a 121-point eyepiece graticule (Leica Microsystems, Wetzlar, Germany).
In brief, cross sections of testes from 4 to 6 MHAR KO or WT mice at d12, 21, 50, and 100 were examined using a Leitz 363 plan apo objective lens (×63) fitted to a Leitz Laborlux microscope and a 121-point eyepiece graticule (Leica Microsystems, Wetzlar, Germany).
Then a 25 point Chalkley eyepiece graticule fixed in one of the eyepieces of the microscope was applied to each vascular hotspot at ×200 magnification [Chalkley grid area of 0.1886 mm (Nikon microscope, Eclipse E400)] in such a way that maximum number of black dots in Chalkley graticule fell on or within immunostained microvessels.
A 25-point Chalkley eyepiece graticule was applied to each hot spot and oriented to permit the maximum number of points to hit on, or within the areas of immunohistochemically highlighted microvessel using × 200 magnification.
On a higher magnification (× 10 ocular and × 20 objective, Chalkley grid area 0.22 mm), a 25-point Chalkley eyepiece graticule was applied to each hot spot and oriented to permit the maximum number of points to hit on or in an immunostained microvessel.
Morphometric analysis was performed with a light microscope (Nikon, E200, City, Japan), with an integrating eyepiece (composed of 100 points and 50 lines) using a point-counting technique [ 13, 14, 29].
On a higher magnification (× 10 ocular and × 20 objective), a 25-point Chalkley eyepiece graticule (Chalkley grid area 0.22 mm) was applied to each hot spot and orientated to permit the maximum number of points to hit on or in a microvessel.
