Anti mouse igg conjugated to peroxidase

The serum antibody titer against recombinant GVAC08 containing GnRH, GnRHR, and ZP3 was determined by ELISA. Briefly, microtiter ELISA plates were coated with 200 ng/well of GVAC08 protein and incubated overnight at 4 °C. The plates were washed 3 times with PBS containing 0.05% tween 20, excess buffer removed, and 3% BSA in PBS added to block non-specific binding. The plates were washed 3 times with PBS containing 0.05% tween 20. Serum collected from the immunized mice was diluted and added to the wells and incubated for 1.5 h. The plates were washed 3 times with PBS containing 0.05% tween 20. Anti-mouse IgG conjugated to peroxidase (Jackson Immunoresearch Laboratories) was diluted to 1:50,000 in PBS and added to the wells and incubated at 37 °C for 1 h. The wells were washed and TMB substrate was added. The reaction was stopped with 1N H₂SO₄ and the absorbance read at 450 nm using Epoch microtiter plate spectrophotometer (Biotek Instruments).

1,25D was purchased from Cayman Europe (Tallinn, Estonia), while ATRA from Sigma-Aldrich (St. Louis, MO). Antibodies CD11b-FITC (cat. No. 21279113), CD14-APC (cat. No. 21270146), CD14-PE (cat. No. 21270144), CD3-PE (cat. No. 21620034), CD20-PE (cat. No. 21279204) as well as appropriately labeled isotype controls were from ImmunoTools (Friesoythe, Germany). Mouse monoclonal anti-POR (cat. No. sc-55477) and rabbit polyclonal anti-actin (cat. No. sc-1616) antibodies were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Goat anti-rabbit IgG and anti-mouse IgG conjugated to peroxidase were from Jackson ImmunoResearch (West Grove, PA).

1,25D was purchased from Cayman Europe (Tallinn, Estonia), ATRA and curcumin were from Sigma. The compounds were dissolved in an absolute ethanol to 1000 × final concentrations, and subsequently diluted in the culture medium to the required concentration. IFNα (cat. no. 11343506), IFNγ (cat. no. 11343536) and antibodies CD11b-FITC (cat. no. 21279113) and CD14-PE (cat. no. 21270144), as well as appropriately labeled isotype controls were from ImmunoTools (Friesoythe, Germany). Mouse monoclonal anti-VDR (sc-13133), anti-Stat1 p84/p91 (sc-464) and anti-p-STAT1 (sc-8394), rabbit polyclonal anti-actin (sc-1616), anti-HDAC1 (sc-7872) and anti-Histone H1 (sc-10806) were from Santa Cruz Biotechnology Inc. (Santa Cruz Biotechnology Inc., CA). Goat anti-rabbit IgG, anti-mouse IgG conjugated to peroxidase, anti-mouse conjugated to biotin and streptavidin conjugated to peroxidase were from Jackson ImmunoResearch (West Grove, PA).