. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. GILL MUSCLE PHARMACOLOGY AND ANATOMY 85 We usually processed whole mounts and sections simul- taneously and therefore followed a schedule designed for whole mounts. All of the steps in this protocol were carried out at 5 °C. • After fixation, rinse four times (1 h for each rinse) in PBS ( M. pH ); or for DA, in M PBS with 1% sodium metabisulfite. • Incubate overnight in blocking solution ( goat serum/1' BSA/PBS): for DA. in- clude \'7c sodium metabisulfite. • Incubate overnight in primary antibody diluted app
. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. GILL MUSCLE PHARMACOLOGY AND ANATOMY 85 We usually processed whole mounts and sections simul- taneously and therefore followed a schedule designed for whole mounts. All of the steps in this protocol were carried out at 5 °C. • After fixation, rinse four times (1 h for each rinse) in PBS ( M. pH ); or for DA, in M PBS with 1% sodium metabisulfite. • Incubate overnight in blocking solution ( goat serum/1' BSA/PBS): for DA. in- clude \'7c sodium metabisulfite. • Incubate overnight in primary antibody diluted appro- priately with PBS. • Rinse four times in PBS (two 30-min rinses, one over- night rinse, one 30-min rinse). • Incubate overnight in secondary antibody, phalloidin. or both, the reagents diluted appropriately in PBS. • Rinse three times in PBS (1 h. overnight. 1 h). • Mount the specimens under coverslips in 60% glyc- erol-1% n-propyl gallate/PBS. Muscle. The branchial muscles were visualized with phalloidin conjugated to the fluorescent probe Alexa Fluor 488 (Molecular Probes. Eugene. Oregon), the conjugate used in a concentration of 1 unit/100 jul in M PBS. For single-stained preparations, whole mounts were fixed for 1 h in 4<7r formaldehyde with M PBS (pH ; 530 mM NaCl). rinsed twice, and then stained overnight. To double- label immunochemically stained preparations, the phalloi- din was added to the tissues at the same time as the sec- ondary antibody. 5HT and YFAFPRQamide. Pieces and sections of demi- branch were fixed overnight in 4% paraformaldehyde in M PBS (pH ; 530 mM NaCl); the fixative was prepared as described in Gainey et al. (1999a). For 5HT, the primary polyclonal antiserum was raised in rabbit to 5HT conjugated to BSA with paraformaldehdye (Diasorin. Still- water, Minnesota). For YFAFPRQamide. the primary poly- clonal antiserum was raised in rabbit to the peptide conju- gated to thyroglobulin (custom synthesis, by Sy
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Keywords: ., bookauthorlilliefrankrat, booksubjectbiology, booksubjectzoology
