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  1. May 2019
    1. The mice were returned to a cage and were kept under a 1 OOW bulb light source to prevent hypothermia. Care was taken to ensure that the eyes are kept covered. The respiratory rate and heart rate were monitored till the mice regained complete consciousness. They were fed ab-limitum post-operatively. Metronidazole (20 mg/kg) was added to the drinking water and the mice were fed this medicated water for 5 days post-operatively. On the th post-operative day, the health of the wound was observed and the surgical clips were removed from the skin
    1. The organic solution of phospholipid was made in a 1:1 mixture of chloroform and methanol at a concentration of 5 mg/ml. To get a thin and uniform lipid film, lipid was dried down onto the walls of a round bottom flask by rotating for 30 min. in a rotary evaporator fitted with a coo!;ng coil and a thermostatically controlled water bath. The temperature of the water bath was maintained 10 °C above the phase transition temperature of the respective phospholipid. The film was stored under vacuum for 1 2 h to remove the traces of the solvent after flushing with nitrogen. Subsequently, lipid film was dispersed in 30 mM Tris-HCI buffer containing O.IM NaCl, pH 7.0 at a concentration of 1 mg/ml of lipid using glass beads of 0.5 mm-3 mm in diameter. Multilamellar vesicles (ML Vs) were obtained by manual swirling for 2 h at room temperature (Bangham et al., 1965). Nitrogen was flushed into the flask and the vesicle suspension was left overnight at 4 °C. The .following day, maintaining the temperature 10 °C abcve the phase transition temperature of the corresponding phospholipid, MLVs were sonicated in a water bath sonifier (Branson 321 0) for 45 min. to get the small unilamellar vesicles (SUVs) (Huang C, 1969).
    1. for 3 hours at room temperature or OIN at 4 °c. The excess antibody was washed by washing the filters with PBST for 15 minutes with at least three changes. The filters were subsequently incubated in the appropriate dilution of the second antibody for one hour at room temperature. Dilutions of the primary as well as secondary antibody were made in 3 % BSA in PBST. Following incubation with the second antibody, the filters were washed vigorously with PBST for 5 minutes. The washing was repeated 5-6 times. Finally, the filters were washed twice in PBS and colour developed with DAB ( 0.5 mg I ml in PBS ) containing 6 ul I 10 ul of 30 % H2o2. The colour reaction was stopped after 5 -10 minutes by washing the filters with distilled water.
    2. apparatus. At the end of the run, the gel was equilibrated for 15 minutes in the transfer buffer ( 25 mM Tris base,, 192 mM glycine, 20% V/V methanol). Immunoblotting was performed essentially as described by Towbin et al., 1979 ) . The proteins-were blotted on to nitrocellulose Schleicher and Schuell ) for 16 hours in LKB Transphor apparatus. After the completion of electric transfer of proteins, the nitrocellulose paper was washed with PBS 10 mM sodium phosphate buffer, pH 7.4, 0.9 % saline for 10 minutes at room temperature, with gentle rocking to wash off any adhering traces of acrylamide gel. The filter was then stained with a dilute solution of amide black. This was made by diluting 5 fold with water, a solution of 0. 2 % ami do black containing 45 % v;v methanol and 10 % v;v acetic acid. The staining was done for 5 10 seconds and the filter washed immediately with distilled water. The proteins transferred to the gel could be seen at this stage. The lane containing protein molecular weight standards was cut out and . preserved by drying and storage in dark. The filter could be cut into appropriate lanes at this stage. The filter was destained by repeated washing with PBS containing 0.2 % Tween-20 ( PBST ) . After the filter destained completely, it was incubated with 3 % BSA made in PBST for 1 hour at room temperature. During this and in all the subsequent steps, incubation of the filter was performed on a rocker platform to ensure a uniform treatment. The filters were then incubated in the appropriate dilution of the primary antibody
    3. Electrophoretic separation of protein samples was carried out on 12.5 % SDS-PAGE in a discontinuous system as described by Laemmli ( 1970 ). The samples were reduced by boiling for 3 minutes in sample buffer containing J3-mercaptoethanol. The samples were then centrifuged for 5 minutes at 10,000 rpm to pellet down all particulate matter, prior to loading on the gel. Electrophoresis was carried out at 100 volts in a LKB vertical slab gel electrophoresis