The mixture was stirred until the entire dissolved and homogeneous. The materials were ready each put in a bottle 100 mL size vial, then sterilized with autoclave for 15 minutes at 121 °C. The preparations hydrogel done within aseptic at LAF cabinet. Evaluation of chloramphenicol hydrogel ophthalmic preparations Organoleptic test Organoleptic hydrogel checked by observing changes in color, odor and clarity. Clarity was checked visually by examination
In 10 g dried sediment sample added 7 ml 0.2 M NH4Cl solution. A mixture of 100 ml hexane: acetone (1:1) was used as a solvent to extract pesticides with overnight shaking for 12 h on reciprocal or wrist action shaker at 180 rpm. The extract was carefully decanted through activated florisil column (2-3 cm), giving twice wash with25 ml hexane: acetone (1:1) to the sediments. The elute was then washed with 200 ml water and then again aqueous layer was extracted with 50 ml hexane. Finally the hexane layer was washed with 100 ml water and then evaporated to dryness with a vacuum rotary evaporator.
The crude enzyme was precipitated at 70% saturation of ammonium sulfate, and the protein was collected by centrifugation (10,000×g for 10 min). It was suspended in minimal volume of double-distilled water. The precipitate was dialyzed and used for enzyme purification using column chromatography. The dialyzed sample was loaded on DEAE-cellulose column, which was equilibrated previously with the buffer A (50 mM sodium phosphate buffer, pH 7.4). The column was washed with the five bed volumes of buffer A, and the enzyme was eluted with buffer A
In the round-bottom flask (100 mL), we placed p-aminobenzoic acid (1.2 g) and ethanol (12 mL). We swirled the mixture until the solid dissolved completely. We used Pasteur pipet to add concentrated sulfuric acid (1.0 mL) to the flask. We added boiling stone and assembled the reflux. Then, we did reflux for 75 minutes.
Apparatus- Chromatography column: C18 (10 microns particle size), with Guard column Flow rate: 1.2ml/min Pressure: 30-40kgf Wavelength: 326nm Mobile phase: methanol : water (95:5 v/v) Internal standard: retinyl acetate Injection volume: 20µl Procedure for Retinol extraction from serum samples- 1) 100 µl of serum sample and 100 µl of Retinyl acetate were added into 12 X 100mm glass test tubes. Vortex-mixed for 30 seconds. Then, kept them at 4 C for 5 mins. 2) 1mL of hexane was added and vortex-mixed intermittently for 60 sec. 3) Centrifuged at 2500 rpm for 12 mins.
The chemicals and dialysis membrane matrix used for the partial purification were ammonium Sulfate, Disodium Hydrogen Phosphate, Sodium Hydrogen Phosphate, Sodium Chloride, dialysis membrane cut off 5 kDa and DEAE-Cellulose. All were obtained from Himedia Labs. 3.9.1 Ammonium sulfate fractionation Aqueous extracts of the M. oleifera were precipitated with solid ammonium sulfate (NH2SO4) to isolate the proteins into different fractions, using varying degrees of saturation. Increasing degrees of saturation with ammonium sulfate were used. Saturation ranges of 0-30%, 30-50%, and 50-80% were initially used for aqueous
Embedding was done with two changes of molten paraffin for 30 min to remove xylene. They were mounted in L blocks with molten paraffin and solidified. The solidified blocks were trimmed to small size and sectioned using microtome (Spencer U.S.A) 3 to 5-micron thickness. 5.2.3 Toluidine blue Staining: Toluidine blue is a metachromatic stain that stains nucleus which appears blue in color. The isolated rat's brain was fixed and processed as per the tissue processing procedure, and then brain tissue slices were prepared for toluidine blue staining.
Then, the test media is then incubated at 37 ° C, for 18-24 hours. Rinsing reusable instruments The samples were rinsed with 40 ml of pyrogen-free water using a glass beaker that is free from pyrogens. Endotoxin testing using STV A total of 0,2mL from the water obtained from the rinsing was placed in the STV containing LAL reagent and was shaken for 20 to 30 seconds. Then STV was placed in an incubator at 37 ° C for 60 ± 2 minutes. STV was then observed by reversing the reaction tube in one smooth motion.
While, 5µl of purified PCR product, 2µl buffer R, 1µl BamHI, 1µl HindIII and 11µl distilled water were added into SOD gene tube. Next, 1 µl of 0.2mg/ml RNase was put inside both tubes and spinned by using microcentrifuge for a few seconds. Those tubes were then placed into 37ºC water bath and incubated for 2 hours. The tubes were deactivated by heat at 65ºC for 10 minutes and stored in -20ºC. Results Measurement of DNA concentration Equipment Optical
The first one was 1X TBS-T (Tris Buffered Saline) that contains 50 mM Tris (pH 7.5 at RT, pH 8 at +4oC), 150 mM NaCl, 0,1% (v/v) Tween-20. The secon one was the blocking Solution that contains 5% non-fat milk powder dissolved in TBS-T. The third ones were primary and secondary antibody. Also, Antibody dilution is done between 1/1000-1/10000 in 5% non-fat milk dissolved in TBS-T. The last one was the chemiluminescent substrates whose Luminol and peroxide solutions are mixed at same volume to prepare working solution.