WB
Western blot analysis of IL-1 beta on different lysates with Mouse anti-IL-1 beta antibody at 1/1,000 dilution.
Lane 1: THP-1 cell lysate
Lane 2: THP-1 treated with 80nM TPA overnight then replaced with 100ng/mL LPS for 6 hours add 300ng/mL BFA for last 3 hours cell lysate
Lysates/proteins at 10 µg/Lane.
Exposure time: 3 minutes;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Mouse IgG - HRP Secondary Antibody at 1/50,000 dilution was used for 1 hour at room temperature.
ICC/IF
Immunocytochemistry analysis of THP-1 cells treated with 80nM TPA overnight then replaced with 100ng/mL LPS for 6 hours add 300ng/mL BFA for last 3 hours labeling IL-1 beta with Mouse anti-IL-1 beta antibody at 1/100 dilution.
Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Mouse anti-IL-1 beta antibody at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Mouse IgG H&L (iFluor™ 488) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.
beta Tubulin (red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Rabbit IgG H&L (iFluor™ 594) were used as the secondary antibody at 1/1,000 dilution.