Th 4 100 inverted microscope

The sections (35 μm) were washed in 0.1 M neutral phosphate
buffer, mounted on slides and air dried on a slide warmer at 50° C. The
sections were immersed in 1% sodium hydroxide in 80% alcohol,
followed by 70% alcohol and in distilled water. The sections were
transferred to a solution of 0.06% potassium permanganate, rinsed in
distilled water and placed in staining solution (0.0004% solution of
Fluoro-Jade B in 0.1 % acetic acid). The sections were washed in
distilled water for one minute three times and air dried on a slide warmer at 50
degrees C. The dried sections were cleared by immersion in xylene and cover
slipped with DPX.
Images were taken in six areas of interest on the left and right dorsal
and lateral cortices and left and right CA1 regions of the hippocampus, using
10× objective of an Olympus TH 4-100 inverted microscope (supplementary Fig. 1).
These images at 10X were used for blinded quantitation of the
number of dead neurons in one coronal section of the rat brain at the level of
the hippocampus . The total sum and average number of stained dead neurons were
counted on each of the 10X sections.

Myocardial fragments were stained by Masson's trichrome for visualization and quantification of fibrotic tissue. Light microscopy was performed using an Olympus TH4-100 inverted microscope that was connected to an Olympus Microfire Digital Camera (New York, NY). For quantification of fibrotic tissue, pictures from each heart section (thickness of 3 μm) were taken systematically to ensure that the entire extent of the tissue section had been covered. Five different hearts were used per group, and a total of 20 sections were analyzed using the Image Pro-plus software (Media Cybernetics, Silver Springs MD). The percentage of fibrotic tissue in each micrograph was measured and averaged per heart. The average value for each heart was then utilized for statistical analysis. Vascularization Index (number of vessels/mm²) was determined in endothelin-1 immunostained sections of the left ventricle of mice from all groups. The number of capillaries per area was determined in 50 random fields per group, following the “Forbidden Line Principle” [13 (link)].