ON TESTIS AND EPlDlDYMlS OF RATS - Pondicherry University ...

3.17 Preparation of tiasue humogenates of cpididymia
Epididymis was washed several times in normal saline in order to remove
maximum number of sperm attached to the epididymal parenchyma. The caput,
corpus and cauda regions of the epididymides were separated and homogenized
separately in cold normal saline with the help of glass teflon homogenizer. The
homogenates were centrifuged at 800 g for 20 min at 4°C. The supernatants were
collected and used for various biochemical analyses.
3.18 Preparation of tissue homogenate of kidney
Kidney was homogenized in normal saline with the help of glass teflon
homogenizer. The homogenate was centrifuged in a refrigerated centrifuge at the
speed of 800 g for 20 min at 4'C. The supernatant was used for the biochemical
analyses.
3.19 Determination of free radicals/ reactive oxygen species in tissues
Production of free radicals such as superoxide anion, nitric oxide and
hydrogen peroxide were determined by the standard methods as described below.
3.19.1 Determination ofsuperoxidc anion
Superoxide anion production was measured by the method of Prodczasy and
Wei (1988) which is based on the rrduction of iodonitrotetrazolium violet (INT). The
reaction mixtures contained 750 pL of the tissue homogenate and equal volumes of
4.9 mM INT, 0.3 mM EDTA and 0.92 mM sodium carbonate (see Appendix 1.14).
The pH of the mixture was adjusted to 10.2. Blank was prepared simultaneously by
adding all the reagents except the tissue extract. The reaction mixture was incubated
for 15 min at room temperature. The reaction was terminated by placing the tubes in
a boiling water bath for 1 min and then cooled to room temperature. After cooling
absorbance was measured at 505 nrn against blank.