Polyclonal rabbit anti human ttr antibody

After iPS-MLs or SH-SY5Y cells were cultured with the appropriate TTRs for 3 days, culture supernatants were mixed with sample buffer (Bio-Rad Laboratories, Hercules, CA, USA) containing 2-mercoptoetanol, and boiled at 95°C for 5 min. Resultant samples were separated by SDS-PAGE and transferred to PVDF membranes (Bio-Rad). Polyclonal rabbit anti-human TTR antibody (Dako; diluted 1:1000) was used as the primary antibody, and polyclonal goat anti-rabbit antibody conjugated with HRP (Dako; diluted 1:5000) as the secondary antibody. The reaction was visualized using ECL prime reagents (GE Healthcare, WI, USA), and detected using a LAS-4000EPUVmini (GE Healthcare).

To stain CD14⁺ monocytes, iPS-MLs and SH-SY5Y cells were cultured with the above-described TTRs, and re-suspended at a density of 2 × 10⁵ cells/mL in DMEM medium (Gibco). Next, cells were adhered to slide glass using Cytospin, and fixed in 4% formalin phosphate buffer. After proteolytic activation for 10 min by proteinase K (Dako), samples were treated with methanol containing 0.3% H₂O₂ for 20 min to remove endogenous peroxidase activity. PBS containing 5% skimmed milk was used to block nonspecific background staining. Polyclonal rabbit anti-human TTR antibody (Dako) was diluted (1:100) in Dako REAL Antibody Diluent (Dako), and used as the primary antibody. Envision System-HRP Labelled Polymer Anti-Rabbit (Dako) was used as the secondary antibody. The reaction was visualized using the Dako Envision + kit/HRP (DAB) (Dako). TTR⁺ cells in CD14⁺ monocytes, iPS-MLs and SH-SY5Y cells were counted. Briefly, five visual fields were randomly chosen from each stained section, and the TTR⁺ cell number in each visual field determined. The average number of TTR⁺ cells per visual field was calculated.

After culturing iPS-MLs or SH-SY5Y cells with the appropriate TTRs for 3 days, TTR concentration in each culture supernatant was measured by ELISA. Briefly, 96-well plates were coated overnight at 4°C with supernatants collected in carbonate buffer. The next day, plates were blocked with coating buffer containing 0.5% gelatin for 1 h at room temperature. To detect TTR, polyclonal rabbit anti-human TTR antibody (Dako; diluted 1:1000) was used as the primary antibody, and polyclonal goat anti-rabbit IgG antibody conjugated with HRP (Dako; diluted 1:5000) as the secondary antibody, which were sequentially incubated for 1 h at room temperature. After incubation with SureBlue™ TMB Microwell Peroxidase Substrate (KPL, Gaithersburg, MD, USA), absorbance was detected at 450 nm.