Sentence examples for blocking buffer were used from inspiring English sources
Alexa Fluor 488-labeled goat anti-rabbit IgG (Invitrogen/Molecular Probes, Eugene, OR) diluted 1∶2000 and fluorescent nucleic acid stain, 4'6-diamidino-2-phenyl-indole dihydrochloride (DAPI) (Invitrogen/Molecular Probes) diluted to a final concentration of 0.25 µg/ml in blocking buffer were used to detect antibody binding and the presence of spirochetes, respectively.
Alexa fluor 647 conjugated streptavidin (1∶100 in blocking buffer) was used to detect 5-BP crosslinked to tissue sections, and Alexa fluor 488 anti-rabbit IgG and Alexa fluor 568 anti-mouse IgG secondary antibodies (1 1000 in blocking buffer) were used to detect primary antibody staining.
Anti-Bak (Sigma, B-5897) or anti-P-eIF2 α (Cell Signaling, Hitchin, UK; S 3398) primary antibodies and HRP-donkey anti-rabbit secondary antibodies (GE Healthcare, Little Chalfont, UK) in blocking buffer were used to detect N-Bak or P-eIF2a, respectively.
Protein extraction and western blotting were performed as described previously [ 27] Anti-lipocalin-2 goat polyclonal antibody (R&D Systems, Inc., AF1857) diluted 1 250 and anti-β-actin goat polyclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) diluted 1 500 in blocking buffer were used as primary antibodies.
GFP-Dcp1 α-overexpressing cells were fixed with 4% PFA, 5 mM MgCl2 in PBS for 15 min at RT, washed two times with 1 × PBS/5 mM MgCl2 and treated as in situ hybridized samples except that mouse monoclonal anti-DCP1 α antibodies in the blocking buffer were used and signal was not amplified.
The washes were performed between each step and the blocking buffer was used for dilution of each of the reagents that were overlaid onto the tissue sections.
For both primary antibodies, goat anti-rabbit immunoglobulin G conjugated with horseradish peroxidase (Pierce; Product Code: 31463); 1∶10000 in blocking buffer) was used as the second step antibody, and ChemiGlow reagent (Alpha Innotech) was used as a substrate.
Rabbit-anti-CBM (diluted 1 3,000 in blocking buffer) was used as the primary antibody for detection of the interaction.
Secondary anti-goat-horseradish peroxidase conjugate (Pierce Biotechnology) in blocking buffer was used at 1 10000 for Myostatin for 1 h.
Rabbit anti-xylanase T-6 antibody (diluted 1 10,000 in blocking buffer) was used as primary antibody for detection of the interaction.
