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

1.7.3 Estradiol controls
Two vials. 1 mL each, Levels I and 11, contained low and high conccntrulions
of esuadiol in human serum with a non-mercury preservative.
1.7.4 Estradiol-Biotin Conjugate concentrate
One vial, 0.3 mL, contained biotinylated estradiol in a protein-based (BSA)
buffer with a non-mercury preservative. Diluted prior to use with the
estradiol -biotin conjugate diluent.
1.7.5 Tetramethylbenzidine Chromogen solution (TMB)
One amber bonle, 11 mL contained a solution of TMB in citrate buffer with
hydrogen peroxide.
1.7.6 Wash concentrate
One bottle, 60 mL contained buffered saline with a nonionic detergent. Diluted
25-fold with deionized water prior to use.
1.7.7 Stopping solution
One vial, 11 mL contained 0.2 M sulfuric acid
1.8 3P-Hydroxystcroid dchydrugcnasc
1.36 g of tris was dissolved in 100 mL of distilled water (Solution A)
0.9 mL of concentrated hydrochloric acid was made up to 100 rnL with
distilled water (Solution B).
62.5 mL of solution A and 55.25 mL of solution B were mixed and made up to
250 mL with distilled water. The pH was adjusted to 7.2 with 1 N sodium
hydroxide.
1.8.2 Sodium pyrophosphate buffer (100 pM; pH 9.0)
13.3 mg of tetrasodium pyrophosphntc was dissolvcd and made up to 500 1111~
with distilled water and pH was adjusted to 9.0.
1.8.3 NAD (0.5 pM)
1.66 mg of NAD was dissolved in 5 mL of distilled water.