To deal with both dark and light text scenarios, the original SWT implementation runs the whole text detection flowchart twice – in gradient and counter-gradient directions – and merges the results of both directions.
Paraffin sections were de-paraffinated in xylene for 10 min twice, a gradient of alcohol (100%, 95%, 85%) for 10 min, and were rinsed in deionized water for 5 min. After washing with 2 mg/ml glycine solution diluted in double distilled water for 10 min, the sections were permeablized with 0.5% Triton X-100 in PBS for 20 min, and then washed twice with PBS for 10 min each rinse.
For SEM, anthers at various developmental stages were pre-fixed with 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.2) for 24 h, dehydrated twice using a gradient ethanol serial (30%-50%-70%-85%-100-100%), then replaced ethanol with isopentyl acetate for 20 min. After that, samples were dried with critical-point drying method then sputtered coating with gold.
The virus was grown in HEK293 cells and purified by banding twice on CsCl gradients, then dialyzed and stored at −20°C.
Briefly, adenoviral lysates from twenty-four 150 mm plates were banded twice on CsCl gradients and desalted twice with a PD-10 size exclusion column (Amersham Scientific, Piscataway, NJ) into HEPES buffered saline (HBS; 21 mM HEPES, 140 mM NaCl, 5 mM KCl, 0.75 mM Na2HPO4·2H2O, and 0.1% (w/v) dextrose; adjust pH with NaOH to 7.5; and filter sterilize) containing 5% glycerol, and stored at -70°C.
It is based on comparisons made by Clement Ley, Sprung, Koppen, and others, and is that the number expressing the wind force on the Beaufort scale shall be twice that expressing the gradient in millimetres of mercury per degree (111 km.).
The recombinant virus vectors were grown in HEK-293 cells and twice purified by CsCl gradient centrifugation, and titers were determined by serial dilution end point assay.
The results show that NoV GII-4 VLPs purified twice by sucrose density gradient centrifugation followed by ultrafiltration maintain their icosahedral capsid structure and their capacity to bind HBGAs.
After another two washes in PBS, the samples were dehydrated with ethanol gradient, replaced twice with propylene oxide, soaked in ethoxyline resin over night, and mounted at 60°C for 48 hours.
After 5 days in culture, blast cells were separated by a discontinuous Percoll (Sigma) gradient, washed twice and adjusted to 10 cells/200 μl in cold phosphate-buffered saline (PBS) containing 0·01% of sodium azide and 10% of fetal calf serum.
