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Protocol (a) Turn on spectrophotometric plate reader in time to have it properly warmed up when samples are ready to read (b) Label a 12 75-mm tube for each tissue culture dish or well of multiwell plates to be analyzed Add a volume of 1 M NaOH equal to the volume of substrate used (See step (d)) to each tube (c) Rinse cells twice with TBSS (d) Add an appropriate volume of substrate solution to tissue culture dishes or wells (1 ml per 35-cm dish, 05 ml per well of 12-well plate, etc) Time the addition of substrate so that cells in each well or dish are in contact with the solution for the same length of time.



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background-color: #fff; /* Make sure the bg is white */

(e) Incubate for 5 to 15 min, depending on the rate of the reaction (as seen by the density of yellow developing in the substrate; the ultimate OD when measured must t within the linear range of the standard curve) (f) Transfer the substrate solution to the NaOH-containing tubes set up earlier Do this in the same sequence in which substrate was added to the dishes or wells, and remove substrate so that each dish or well is exposed to the substrate for the same length of time (g) If cultures are to be used for additional assays (alkaline phosphatase cytochemistry, DNA, etc) add TBSS to the cells immediately, and do additional rinses and xation as soon as time permits (h) Set up and label a 96-well plate.





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Figure 9.17 (a) Gradient of the Bragg mismatch with respect to wavelength @Dk=@ l as a function of the Bragg matched grating half-angle u0. The gradient is evaluated at the Braggmatched angle and wavelength; (b) gradient of the Bragg mismatch with respect to angle @Dk=@ u versus u0.

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i) To the rst row add 200 l of a mixture of equal parts of 1 M NaOH and substrate buffer to serve as blanks ii) Transfer 200 l of appropriately diluted (or undiluted) experimental samples to corresponding labeled wells (usually 4 replicate wells per sample) As a rough guide for determining whether samples require dilution, compare the intensity of color of the samples to that of the highest concentration of p-nitrophenol Dilutions are made with the same solution used for blanks iii) Set up a standard curve by transferring 200 l diluted p-nitrophenol standards to wells (See Section 23) (i) Read the absorbance at 405 nm on a microplate reader Do further dilutions for samples that give readings beyond the linear range of the standard curve and read samples again Note any dilutions made..

ovec(X ) -I ,-I , >l X)' = det(X)[(vec X )(vec X ) uvec( (6) X (m x m) symmetric nonsingular:

In order to improve the exibility and e ciency of BSS/ICA schemes, we can use multistage sequential or multilayer neural networks. Fig. 7.6 shows various possible con gurations of multilayer neural networks. In this way, we can improve dramatically exibility and performance of blind separation or extraction by applying di erent algorithms or di erent activation functions in each layer. Furthermore, this allows us to combine SOS and HOS statistics algorithms to extract at various layers various sources with di erent statistics properties. The layers should work rather sequentially, one by one, i.e., after achieving convergence in the rst layer the second layer will start to work, and so on.

FIGURE 8.2 Converting layers into a Smart Object. Select the layers (left), and choose Layer Smart Objects Convert to Smart Object. The Smart Object replaces the layers in the Layers palette (right).

Running this will simply print out a list of the labels you are using right now. That s useful, but you can extend it a little bit. Listing 11-2 does the same thing, but allows you to select a label, whereupon it prints all the messages labeled thusly. Have a look at the listing, and then you ll walk through the clever bit. Listing 11-2: Retrieving the Messages from a Certain Label

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