One step nbt bcip

TMB peroxidase substrate and stop solution were purchased from Kirkegaard & Perry Laboratories, Inc. (Gaithersburg, MD, USA). One-Step NBT/BCIP (nitro-blue tetrazolium and 5-bromo-4-chloro-3'-indolyphosphate) was purchased from Thermo Fisher Scientific Inc. (Rockford, IL, USA). ZyMAX™ horseradish peroxide (HRP)-labeled goat polyclonal anti-human IgG (H + L) was purchased from Life technologies (Camarillo, CA, USA). Human papillomavirus vaccines, Cervarix^® (including recombinant human HPV-16 and -18 L1 proteins; GlaxoSmithKline Biologicals, Rixensart, Belgium) and Gardasil^® (including recombinant HPV- 6, -11, -16, and -18 L1 proteins; Merck, White Station, NJ, USA), were purchased from the manufacturers. World Health Organization (WHO) international standard HPV-16 (NIBSC code: 05/134, 5 units per ampoule) and HPV-18 antibodies (NIBSC code: 10/134, 8 units per ampoule) were purchased from NIBSC (London, UK). Normal anonymized human female sera, which were collected under an IRB approved protocol for de-identified, were purchased from Complex Antibodies, Inc. (Margate, FL, USA) and were used as a control serum which showed no immunoreactivity against the Cervarix^® antigen based on our prior ELISA.

One-eighth of the WT UFEs and one ChgH ko UFE were loaded onto a 12.5% polyacrylamide gel. The gel was stained by Coomassie Brilliant Blue-250.
One-fortieth of the WT UFEs and half of the ChgH ko UFEs were loaded onto a 12.5% polyacrylamide gel. After SDS-PAGE, proteins were transferred to a polyvinylidene fluoride membrane. The membranes were blocked in TBS containing 1% bovine serum albumin and 0.05% Tween-20 and then incubated with primary antibodies (Table S6). After washing, the membranes were incubated with secondary antibodies (Goat anti-mouse (or -rabbit) IgG alkaline phosphatase (AP) conjugate (Sigma-Aldrich) at 1:2500 dilution, and the alkaline phosphatase activity was visualized by OneStep-NBT/BCIP (Thermo Fisher Scientific Inc).

Cells were rinsed in PBS, fixed in 4% paraformaldehyde for 20 minutes at room temperature, washed with PBS, and then stained with One-Step NBT-BCIP (catalog no. 34042; Thermo Scientific) for 20 minutes, rinsed with PBS, and air dried.