After a final washing with PBS, grayscale signals from each secondary antibody were captured with a DS-QiMc camountedunted on an 80i epifluorescence microscope (both from Nikon, Tokyo, Japan), and the resulting monochrome images were merged with NIS-Elements D image analysis software (Nikon).
However, as the market has grown in terms of both buyers and sellers, private companies have mandated an increasing number of restrictions, with each handling secondary share sales differently.
To simplify the design of the windings, graphics are generated to calculate the turn-ratio and the polarity of each secondary winding, with respect to the primary winding.
As a result, by finding configurations (i.e., specifying channel allocations and power control at each secondary link) with minimum MRF system energy, we are able to both maximize the offered capacity per link and provide to SUs an efficient scheduling scheme avoiding collisions and, hence, improve the overall secondary network performance.
After four washes of 10 min each with PBS, secondary reagents were applied for 30 min at room temperature.
We then fit a series of multivariate models each with one secondary explanatory variable removed, always noting the value of the β coefficient associated with incarceration.
Briefly, slides were blocked with horse serum (5 min), incubated in a humid chamber for 60 min with primary antibody, and for 30 min each with biotinylated secondary antibody and avidin-biotinylated horseradish peroxidase complex (ABC).
The slides were then incubated for 50 min each with the secondary antibody (Biotinylated Link, Dako Japan) and peroxidase (STREPTOAVIDIN-HRP, Dako Japan), followed by visualization with 3,3′-diaminobenzine tetrahydrochloride (DAB + CHROMOGEN, Dako Japan).
After noting the value of the regression coefficient associated with physician experience in the full model, we used a manual stepwise approach to fit a series of reduced models, each with one secondary explanatory variable dropped from the full set.
Cells were washed four times with PBS at RT for 5 min each, incubated with secondary fluorescent antibodies at 1 400 dilution in PBS with 1% BSA and 0.05% saponin for 60 min, re-washed with PBS, and then mounted with Fluoro-Gel anti-fade mediumng mEMSum (EMS) for imaging.
