Modulatory Role for Substance P: MATERIALS AND METHODS(5)

MATERIALS AND METHODS(5) Plates were then incubated for 2 h at 37°C. 5) Secondary antiserum. Two hundred microliters of goat anti-rabbit-immunoglobulin G-biotin conjugate (IgG-B; Sigma) diluted 1:1000 in assay buffer was added and incubated for 1 h at 37°C. 6) Avidin-Biotin Peroxidase Complex (ABC). ABC (200 |xl; Vector Laboratories, Burlingame, CA) diluted 1:200 in assay buffer was added and incubated at 37°C for 20 min. ABC solution was prepared 20-60 min before use. 7) Enzyme reaction. Two hundred microliters of peroxidase substrate (0.015% H202, 0.125% ortho-phenyle-nediamine [OPD; Sigma] in 0.1 M citrate-phosphate buffer, pH 5.0) was added and incubated for 20 min at room temperature in the dark. The solution was prepared immediately before use.
The reaction was stopped by adding 50 |xl of 2 M sulfuric acid. After 20 min, the optical density was measured at 490 nm by using a plate reader (Mios Merck, Darmstadt, Germany), and the results were entered into a PC computer for mathematical processing and statistical analysis using routines written for the computer program SigmaPlot (Jandel Scientific Co., Corte Madera, CA). A standard curve was obtained by plotting the B/Bo ratio against the logarithm of the corresponding dose, in which В was the mean absorbance value for each pLH dose minus the mean of the 0% values (blank wells) and Bo was the mean absorbance of the 100% values (wells with only specific antiserum added) minus the mean absorbance of the 0% values. The curve was then adjusted to a logistic four-parameter equation using the SigmaPlot program, which generated the four parameters by least-squares regression; and the resulting equation was used to obtain the concentration of a given sample from its absorbance value. Buy Asthma Inhalers Online
Data Analysis Differences in hormone release and content were analyzed using the general linear model of SigmaStat (Jandel Scientific Co.). Before comparison, pairs of data groups were tested for Normality and Equality of Variance. If data groups passed both tests, a Student’s Mest was applied. Otherwise, pairs of groups were compared with a Mann-Whitney Rank Sum test. A one-way analysis of variance followed by Student-Newman-Keuls test was applied to compare multiple groups. Differences were considered significant at p < 0.05.

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