Total protein extract was resolved by SDS polyacrylamide gel electrophoresis (15% polyacrylamide) and electroblotted from the gel onto a PVDF membrane using Tris glycine buffer and then blocked overnight in 5% (w/v) skim milk solution.
Cross sections of human aortic atherosclerotic plaque were deparaffinized, dehydrated, and permeabilized with 0.05% Tween in citrate buffer, and then blocked with normal horse serum.
ELISA plates were coated with TNP-BSA overnight in a sodium carbonate buffer and then blocked with 0.5% BSA in PBS for one hour at room temperature.
Briefly, antigen retrieval was achieved in citrate buffer, and then blocked, followed by incubation with rabbit monoclonal Ki67 antibody (Thermo Fisher Scientific Fremont, CA).
After incubation, we washed the plate two times with 150 μL of PBS buffer with 0.1% Tween-20 (PBST buffer) and then blocked each well with 100 μL PBST plus 1% BSA for 1 h as described in ref (18).
Twenty hours after coating, wells were washed with HANKS buffer, and then blocked with HANKS containing 1 mM MgCl2 and 1 mM CaCl2 (HANKS+) and 1% BSA for 1 hr at room temperature.
Following SDS-PAGE separation, protein bands were transferred to a nitrocellulose membrane in buffer containing 1× NuPAGE transfer buffer, 20% methanol, and then blocked in 5% nonfat dry milk with 1×TBS including 0.05% (v/v) TWEEN for 1 hour.
Briefly, after deparaffinization, sections were subjected to epitope retrieval in tris-HCl buffer (pH 9.0) and then blocked in 3% hydrogen peroxide for 10 minutes.
Briefly, microtiter plates (Nunc, Roskilde, Denmark) were coated overnight at 4°C with 45 μg/ml BSA diluted in 0.1 M carbonate-bicarbonate buffer (pH 9.6) and then blocked for 1 h at 37°C with PBS containing 0.2% Tween 20 (0.2% PBST) after washing with 0.05% PBST.
In brief, 96-well Nunc Maxisorp immunoplates (VWR International AB, Stockholm, Sweden) were coated overnight with antigen (Finnish Fur Breeder´s association, Vaasa, Finland) diluted 1 1500 in coating buffer (50 mM NaHCO3 buffer, pH 9.6, SVA, Uppsala, Sweden) and then blocked with blocking buffer (PBS containing 1% BSA, SVA).
SPOT membranes were washed with TBS (50 mM Tris-buffer saline, pH 7.0) and then blocked with TBS-CT (Tris-buffer saline, 3% casein, 0.1% Tween 20, pH 7.0) at room temperature under agitation or overnight at 4°C.
