APRIL (human), mAb (Aprily-8)

Figure 2: FACS analysis of cells with MAb to APRIL (Aprily-8).Method: HEK 293 cells were mock transfected or transfected with an expression plasmid coding for a non-cleavable human APRIL. Cells (5x10⁵) were incubated on ice for 30 min. in 50µl FACS buffer (PBS, 5% fetal calf serum, 0.02% azide) containing 10µg/ml of Aprily-8 antibody. After washing in FACS buffer, FITC-conjugated antibody to mouse IgG was added. Cells were incubated on ice for 30 min., washed and analyzed by flow cytometry.

Figure 3: Immunostaining of HEK 293 cells transfected with a human APRIL expression plasmid (left panels), or mock transfected (right panel) by Aprily-8.Method: 3 days after transfection of cells with the indicated constructs, cells were fixed with 4% formaldehyde 5 min. at RT. After a wash in PBS, samples were dehydrated by washes in 60%, 80%, 90%, 100% EtOH and xylol. Cells were then dried and embedded in paraffin. Sections were cut, mounted on slides and dried overnight at 50°C. Slides were then successively washed 2x 10 min. in xylol, 2x 10 min. in 100% ethanol, and then treated 10 min. in 100% methanol/0.6% H₂0₂ to inhibit endogenous peroxidase. Samples were rehydrated by washes in 90%, 80%, 60% ethanol and PBS. After micro-wave treatment, slides were washed 3x in PBS, blocked with IgG, and incubated for 1 hour with 5µg/ml Aprily-8 or control mouse IgG (isotype control) in 1%BSA / 1x PBS for 1 hour. After PBS washes, samples were incubated with the secondary Ab for 1 hour, washed in PBS and revealed with StreptABComplex/HRP (Vector) and AEC.