Tissue was washed three times with incubation buffer and then incubated with donkey anti-goat Oregon Green 488 (1 100, Beckton Dickinson) at room temperature for 1 hour.
After 10 min of incubation, cells were washed twice with washing buffer and then incubated with 100 µl PE-streptavidin conjugate or streptavidin-PE-Cy5.5 at a final dilution of 1∶400 dilution (optimized).
Following incubation, the membranes were washed three times in TBST buffer, and then incubated with horseradish peroxidase (HRP -conjugated rabbit IgG (diluted 1:5000) for 1 HRP -conjugated
Following incubation, the plates were washed four consecutive times with wash buffer and then incubated with the prediluted horseradish peroxidase conjugated antibody for 1 hour at room temperature without shaking.
After 60 min of incubation, cells were washed twice with fresh Kreb's Ringer buffer and then incubated with the D. reticulata extract at 250 and 500 μg/ml, and glibenclamide (50 μg/ml) for 60 min.
These cytochrome-immobilized particles were washed with phosphate buffer and then incubated with peroxidase, EDC, and NHS.
Protein A/G beads (Millipore) were washed three times with lysis buffer, and then incubated at 4°C for 4 h with the lysate-antibody complexes.
The native-PAGE was soaked in 20 mM 2,6-DMP (dissolved in 20 mM phosphate buffer ) and then incubated at 50°C until bands began to appear.
Briefly, each embryo was transferred into a PCR tube containing 1 μL lysis buffer, and then incubated at 65°C for 3 h followed by 95°C for 10 min.
Briefly, urine samples were diluted 100-fold with dilution buffer and then incubated for 1 h at room temperature with polyclonal anti-human L-PTGDS antibody immobilized to the surface of the plate wells.
