Body weights were recorded prior to dosing and weekly thereafter. All gross visible signs and symptoms were also recorded. 2.7.3 Histopathological Analysis Representative samples of the liver and kidney were removed from the control and AMPs LR14 (1,000 mg/kg) administered group of animals. The formalin-preserved tissue sections were processed as follows: (1) fixation in 10 % neutral buffered formalin for 1 h, twice; (2) dehydration in graded series of
alcohol (70 % for 30 min, 90 % for 1 h, and two cycles of 100 % for 1 h each); (3) dehydration again with xylene for 1.5 h, twice; and (4) impregnated in molten wax at 65 °C for 2.5 h with two changes. The processed tissues were embedded in paraffin and sectioned (4 μ thickness) and dried on a 70 °C hot plate for 30 min. The tissues were stained using hematoxylin and eosin (H&E) PRI-724 manufacturer stains. The stained tissues were dehydrated with 70 % ethanol followed by 90 % ethanol, placed in two changes of 100 % ethanol for 3 min each, and cleaned with two changes of xylene (3 min each). The morphological changes were monitored through a bright-field microscope (Leica TP1020, Japan). 2.8 Studies on Generation of Immune Response of AMPs LR14 in a Rabbit A purified preparation of the peptide (200 μg/mL)
was used to immunize a rabbit, followed by the booster doses (100 μg/mL) administered at an interval of 4 weeks. AMPs LR14 as an antigen was injected subcutaneously and the rabbit was bled after 4 months. Blood collected from the animal was subjected
to ELISA in order to detect the Selleckchem mTOR inhibitor formation of antibodies. selleck kinase inhibitor Different dilutions (10 ng/mL, 100 ng/mL, 1 μg/mL, 10 μg/mL) of the antigen (purified AMPs LR14) were added to a microtiter plate and kept for incubation at 4 °C overnight. The plate was washed with 0.01 M phosphate buffer pH 7.2. Casein was added to all the wells and incubated at room temperature for 1 h. Casein was removed from the wells and washed with 0.01 M PBS. The plate was tapped gently on a blotting sheet. Next, primary antibodies were added in different dilutions comprising 1/10, 1/100, 1/500, 1/1,000, 1/2,000, PFKL 1/5,000, and 1/10,000. In one set, 1/10 pre-bled antiserum was taken as the control. Washing was done again with PBS three times and the plate was tapped gently every time. Further, secondary antibodies [goat anti-rabbit IgG and horse radish peroxidase (HRP) conjugate] were added and the plates were incubated for 1 h at 37 °C. This was followed by three rounds of washing with PBS. The substrate o-Phenylenediamine (OPD) at a concentration of 10 mg/mL was added to each well and plate was incubated at room temperature for 30 min. Absorbance was read at 490 nm. 2.9 Statistical Analysis The in vitro antiplasmodial experiments were conducted in triplicate and the results represent the mean of two independent experiments. The in vivo toxicity test was performed for n = 5 per group of rats/dose per batch.