Histology New slides were washed with chromic acid followed by rinsing with diluted NaOH and distilled

water and dried in hot air oven. 0.1 ml poly-L-Lysine was spread evenly on each slide and dried well. The slides were stored for 2-3 weeks at room temperature. the endoscopic biopsy specimens were fixed in 10% buffered formalin and 5m thin section were cut From the paraffin embedded blocks and floated in a hot water by containing distilled water. The flattened section where collected on clean poly-L-Lysine coated glass slides and dried overnight. For optimal adhesion the slides were placed in an oven at 60oC for 1 hour. Of the numerous section cut from Bouins fixed paraffin embedded specimen, one section was used for protein hematoxylin/eosin staining and other were used for immunohistochermistry. The section were deparaffinised using xylkene, treated with absolute alcohol, 90% and 70% grades of alcohol and then dipped in water. The slides were stained with hematoxyliun for 10 min followed by washing with running tap water until the section became blue. Then the slides were stained in 1% eosin for 1 minute and washed in running water for 4-5 minute. The section were dehyderated in ascending grades of alcohol, cleared in xylenee and mounted in DPX or permanent mount. The section were visualized and photographed in an Axioskope Carl Zeiss Microscope (Germany) Immunohistochemistry Immunohistochemistry is a trechnique for localization and visualization and antigen in a section by using an antibody specific for the target proteins. The immunohistochemistry procedure consist of tissue preparation antibody incubation and series of detection reaction. The tissue are frozen or fixed, sectioned and attached to slides. The sections are then waxed, treated with a target retrieval solution. Blocked with a protein based blocking solution and then incubated with a primary and corresponding secondary antibody HRP conjugates and substrate. When adequate colour development is seen, then slides are washed in water to stop the reaction counterstained and covered with a mounting medium.(Sambrook et al., 1989) Reagents Xylene 30% hyderogen peroxinde (h2O2) Various percentage of graded alcohol (100%, 90%, 70%, 50%, and 30%) Phosphate buffered saline (1X PBS) (100ml, pH 7.4): 0.8g NaCl, 0.02g KCl, 0.115g Na2HPO4 and 0.02gKH2PO4 were dissolved in distilled water; the pH waws adjusted to pH 7.4 and made upto 100 ml. Blocking solution: 5% BSA in 1X PBS Colour indicator substrate solution: 0.05% DAB and 0.01% h2o2 were dissolved and diluted in 1X PBS DPX and cover slis Procedure

4m paraffin embedded tissue section were dew axed or deparaffinised in two changes of xylene mfor 10-15 minutes each. The slides were then rehydrated in graded ethanol series (100%, 90%, 70%, 50%, and 30%) for 10 minutes each and then washed with double distilled water and 1X PBS for 10 minutes each. The tissue sections were incubated in blocking buffer and were kept inside the moisture chamber for 1 hour to block non-specific endogenous peroxidise activity. The slides were treated with primary antisera at a range of 1:100 dilutions for 16-18 hour at 4oC. after overnight incubation of primary antibody the slides were washed thrice with 1X PBS twice for 5 minutes and then incubated with corresponding secondary antibody (1:200 dilution) at room temperature flowed by washing twice with 1X PBS for 5 min each. Finally the tissue section were developed with colour indicator substrate (DAB) solution for 5-10 minutes at dark condition and the reaction was stopped by washing with 1X PBS. The sections were then rehydrated in series of grade ethanol (30%, 50%, 70%, 90% and 100%), cleared in xylene and mounted in DPX using cover slips. The slides were vied and photographed in an Axioskope, Carl Zeiss Microscope (Germany) Extraction of protein The biopsy specimen were homogenised in lysis buffer (135mM NaCl, 20mM Tris, and 2mM EDTA containing proteases inhibitor tablets (Roche) pH 7.4). The homogenates were centrifuged for 15 min, 7500 rpm at 4oC and the protein content of the supernatant determined by lowrys method (lowey et al 1951) Determination of protein concentration (lowry method) The carboxyl group of protein molecules reacts with copper and potassium of the reagent to give blue colour potassium biuret complex. The complex together with tyrosine and phenolic compound present in the protein reduce the phosphomolybdate of folin reagent to inanity the colour of the solution. Reagent The following regent were used for the estimation of protein by lowry ET al (1957) Solution C (lowrys reagent) : 50ml of solution A (2% sodium carbonate in 0.1 N sodium