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In order to make the emulsion sample optically clear and to avoid the effect of multiple scattering, 0.1 mL of emulsion was diluted with 15 mL of distilled water.
The crude extract from each animal organ was centrifuged at 12,000 rpm for 2 min and the supernatant (0.1 mL) was added into 1.9 mL of emulsion and subsequently incubated for 10 min at 37 °C.
To 1 mL of emulsion sample, 2 mL of chloroform/methanol (2 1, v/v) were added and mixed using a vortex mixer for 3 s to separate the sample into two phases.
In total, 0.1 mL of emulsion was injected at multiple locations at the base of the tail with a 26-gauge needle.
Each mouse was weighed and injected intradermally at the base of the tail with 0.1 mL of emulsion containing 100 μg collagen, using a 1-mL glass tuberculin syringe with a 26-guage needle.
3 Briefly, a preparation of 100 μg of type II collagen (CII) obtained from bovine nasal cartilage and 300 μg of Mycobacterium tuberculosis in 0.1 mL of emulsion was injected subcutaneously into the base of the tail.
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After passing sterility tests, the vaccine was packaged into autoclaved 2 mL bottle with 1 mL volume of emulsion.
Three weeks later, the mice were boosted with another 0.1 ml injection of emulsion at the base of the tail to induce disease.
The emulsion was sonicated with 100 ml of 2.5%% emulsion stabiliser, polyvinyl alcohol (PVA) (Sigma) (2.5 g/100 ml distilled water) at 55%% power for 10 min over ice to form the final w/o/w solution [ 33, 34].
To an aliquot of 5 ml of this emulsion, 0.2 ml of each extract (0.05 0.250 mg/ml) or ascorbic acids were added and mixed well.
To prepare samples for measurement, 0.1 mL of each emulsion was diluted with 100 mL of distilled water.
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Justyna Jupowicz-Kozak
CEO of Professional Science Editing for Scientists @ prosciediting.com