After Al treatments, peanut roots of 99-1507 and ZH2 were firstly washed in running tap water, and then washed twice with ddH2O thoroughly.
The filtered reaction mixture was firstly washed several times with distilled water and the separated organic layer was dried with anhydrous sodium sulphate for 1 h to remove trace water and decompose traces of unreacted peroxides.
The samples were firstly washed with acetone and deionized water and then immersed into H2SO4 and H2O2 solution in a volume ration of 3 1 to remove the organic contaminants on the surface.
Then, the 6-well microplate was firstly washed by 5%(vol/vol) sterile BSA, and was added into 2 aliquots (0.5 ml each) of prepared Arabidopsis mesophyll protoplasts, then, the protoplasts was gently suspended with 1 ml washing and incubation (WI) solution (4 mM MES pH 5.7, 0.5 M mannitol and 20 mM KCl), and was incubated at 22°C.
40 µg of protein from each sample was applied to a 10% zymography gel and electrophoresed constantly at 90 mA for 60 min. Gels were firstly washed twice with washing buffer (2.5% Triton X-100,50 mmol/L Tris –HCl, 5 mmol/L CaCl2, 1 µmol/L ZnCl2, pH 7. 6) for 45 min, followed by a 42 hour incubation in a buffer containing 50 mmol/L Tris-HCl, 5 mmol/CaCl2, 1 µmol/L ZnCl2, 0. 02% Brij-35, pH 7.6.
After five minutes, the staining solution was removed and the cell monolayer was firstly washed by distilled water and subsequently washed thoroughly with PBS to remove the nonspecific background stain.
Sections were firstly washed three times for 30 min each with a high-stringency wash solution (50%formamide/0.5×SSCSC) at 80 °C, and then three times for 10 min at room temperature in 0.5×SSC.
Firstly, when washing the liver through with PBS it is apparent which parts of the wedge were being 'perfused' by that particular vessel, hence the greater the liver area 'perfused' the more favourable the vessel was thought to be and secondly the two vessels should be in different parts of the liver wedge to ensure optimum perfusion of the whole wedge.
The cartridge will firstly be washed 3 times with 3 ml of absolute methanol and 3 ml of ultra-pure water successively before preparation of the columns.
