The phrase "again for three times" is not correct in standard written English.
It can be used when referring to repeating an action three times, but it should be phrased differently for clarity.
Example: "Please repeat the instructions again for three times."
Alternatives: "repeat it three times" or "do it three more times."
After 1.5 h of incubation at 37°C, the cells were washed three times and incubated with the primary detection Ab for 1 h at 37°C, washed again for three times, and incubated with the secondary detection Ab for 1 h at 37°C and washed again.
The membrane was washed again for three times in PBST.
After three washing steps with TBS-T, the membrane was incubated for 1 h with an alkaline phosphate-coupled secondary anti-mouse (1 in 3000 dilution in TBS-T (Chemicon, Hofheim, Germany)), anti-rabbit (1 in 3000 dilution in TBS-T(Chemicon)) or anti-rat (1 in 5000 dilution in TBS-T (Sigma-Aldrich, St Louis, MO, USA) IgG antibody and then washed again for three times in TBS-T.
After incubation with a 1 : 500 dilution of goat anti-mouse IgG DyLigth 594-conjugated antibodies (Jackson ImmunoResearch, Baltimore, PA, USA) and a 1 : 500 dilution of goat anti-rabbit IgG DyLigth 488-conjugated antibodies (Jackson ImmunoResearch) at room temperature for 45 min, coverslips were again washed for three times in PBS.
The DCs were washed again with cold staining buffer for three times, and the cell surface markers were analyzed by flow cytometry.
After the removal of precipitates, the NHS ester of succinic monoester of mPEG5k (NHS-SC-mPEG5k) in THF was precipitated with 50 mL diethyl ether; the purification process was repeated for three times again [7].
Sections were firstly incubated overnight at 4°C with C3dAb (dilution 1 : 1 : 500 Dakopatts A/S, DK-2600 Glostrup, Denmark) and washed in PBS for three times, again, followed by incubating 10 min with a secondary antibody, IgG antibody- (Fab-) HRP polymer which was used according to the manufacturer's protocol.
In the final session, only SS were again presented for ten times in random ITI.
After washed again in PBS for five times, the slides were embedded in 50% (v/v) glycerol.
Then 25 μL of 0.22 U·mL−1 of acetylcholinesterase from electric eel was added and the absorbance was again read every 45 s for eight times.
That number would have to double again and again, four times over, for London to become as bad as Milwaukee.
