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navigation system that Maria is using E-ticketing and they start for home. The amount Maria is automatically debited for the bus ride will depend upon where she gets off. Giovanni is also on his way back. He has met his lift successfully and will arrive home just after Maria and Elisa. The family is grateful to eventually all be heading home, but Maria is a bit annoyed with the mix-up by the garage and plans to complain to the insurance company when she gets home.
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This example uses a Spreadsheet control to create a simple loan payment calculator in a UserForm. The finished product is shown in Figure 15-16. The user can enter loan information into column B, and the monthly payment is calculated (using a formula) and displayed in the bottom right cell.
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Para lm Biotin Blocking System Peroxidase Blocking Reagent Blocking Buffer (See Section 2.7) Washing Buffer (See Section 2.8) DAKO LSAB System containing: biotinylated anti-rabbit Ig antibody, HRPconjugated streptavidin, and chromagen substrate. Protocol (a) Perform Steps (a) through (e) from Protocol 15.4. (b) Transfer coverslips to Para lm to minimize the amount of liquid required to maintain coverage on slips. (c) Incubate with 10 drops Avidin Blocking Agent, from Biotin Blocking System, for 20 min to suppress endogenous avidin. Wash 2 , each with 2 ml PBSA. (d) Similarly, incubate with 10 drops Biotin Blocking Agent, from Biotin Blocking System, for 20 min to suppress endogenous biotin. Wash 2 , each with 2 ml PBSA. (e) Incubate with 12 drops Peroxidase Blocking Reagent for 8 min to quench endogenous peroxidases. Wash 2 , each with 2 ml PBSA. (f) Add 10 drops of Blocking Buffer and incubate for 5 min at room temperature. Use paper tissue to remove excess Blocking Buffer; do not rinse. (g) Prepare 1/100 dilution of rabbit anti-rat albumin antibody in PBSA with 1% BSA. Add 600 l diluted antibody solution to each coverslip. Incubate at 37 C for 90 min, gently rocking every 30 min to ensure even coverage on substrate. (h) Wash 3 with 2 ml Washing Buffer, for 5 10 min each on rocker. (i) Incubate with 10 drops Biotinylated anti-rabbit Ig antibody solution for 20 min at room temperature. Wash 2 with 2 ml Washing Buffer, for 5 10 min each on rocker. (j) Incubate with 10 drops HRP-conjugated streptavidin solution (including, according to manufacturer s instructions, 4 ml UPW, 4 drops buffer concentrate, and 1 drop concentrated HRP-streptavidin) for 20 min at room temperature. Wash 2 , each with 2 ml PBSA. (k) Prepare substrate solution according to manufacturer s instructions: 2 ml substrate buffer, 1 drop H2 O2 , and 1 drop 3-amino-9-ethylcarbazole chromagen substrate in dimethyl formamide. Incubate for 10 min at 37 C. Watch closely after 10 min for color change; do not overdevelop. Wash 2 , each with 2 ml PBSA. (l) Remove coverglass from PBSA and mount. Hold coverglass with tweezers, touch edge to towel, and aspirate at edge to remove excess liquid. Turn coverslip upside-down and add one small drop Vectashield. Slowly sandwich together coverslip and clean glass slide, and seal coverslip edges with clear nail polish. (m) Record images by photomicroscopy.
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