1. 0.1 M glycine buffer: 7.5 g of glycine and 5.85 mg of sodium chloride were dissolved in one litre of distilled water.
2. Buffered substrate: 2.76 g of lithium lactate was dissolved in 125 ml of glycine buffer containing 75 ml of 0.1 N sodium hydroxide solution. This was prepared just before use. 3. 0.4 N NaOH.
4. 5.0 mg of NAD+ was dissolved in 1.0 ml of distilled water just before use.
5. 2,4-dinitrophenyl hydrazine reagent (DNPH) : 200 mg of DNPH was dissolved in one litre of 1 N HCl.
6. Standard pyruvate solution: 12.5 mg of sodium pyruvate was dissolved in 100 ml of buffer. Procedure
To 1.0 ml of the buffered substrate, 0.1 ml of serum was added and the tubes were incubated at 37°C for 15 min. After adding 0.2 ml of NAD+ solution, the incubation was continued for another 15 min. The reaction was then arrested by adding 1.0 ml of DNPH reagent and the tubes were incubated for a further period of 15 min at …show more content…
The slides were blocked using 5% Bovine serum albumin in Tris buffered saline-Tween 20 (TBST) for 1 h at room temperature to avoid non-specific binding. Then, the specific primary antibody for PCNA (IHC grade) and GST-pi (1:800 dilutions) were added to the sections and incubated at 4◦C, overnight. After washing, the slides were incubated with secondary antibody IgG-HRP (1:1000 dilutions) in TBS and 0.05% Tween 20 at room temperature. They were developed with DAB solution containing 0.05% DAB, H2O2 in TBS in a dark room for 10min and counter stained with hematoxylin followed by dehydration and mounted with DPX. Quantitative analysis was made in a blinded manner under a light microscope. Each section was examined at a magnification of 20× and the ratio of positive area of hepatic tissue area was calculated. The mean of five different fields in each section was