In this step, blocking buffer was used in place of serum.
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.
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.
Rabbit-anti-CBM (diluted 1 3,000 in blocking buffer) was used as the primary antibody for detection of the interaction.
Rabbit anti-xylanase T-6 antibody (diluted 1 10,000 in blocking buffer) was used as primary antibody for detection of the interaction.
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.
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.
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.
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.
Three different blocking buffers were used, depending on the primary antibody: human LMNA, 3% donkey serum in PBS; mouse LMNA, 10% rabbit serum in PBS; mH2A and histone H3, 4% bovine serum albumin, 2% donkey serum, 2% rabbit serum in PBS.
