Goat anti mouse igg secondary antibody

Drug‐induced cell surface loss of the μ opioid receptor was determined using an enzyme linked immunosorbent assay (ELISA) (Lowe et al., 2015 (link)). HEK293T cells were transfected as described in Section 2.2 with 3 μg of HA‐tagged μ opioid receptor or 3 μg pcDNA3.0 (empty vector) and incubated for 24 h, with cells then being plated onto poly‐l‐lysine coated 24‐well plates at 250,000 cells per well and incubated for a further 24 h. For experimentation, cells were serum starved for 15 min using Opti‐MEM, then agonist (morphine, DAMGO, fentanyl, carfentanil) or vehicle control was added and incubated for a further 1 h at 37°C. Cells were then fixed using 4% paraformaldehyde for 5 min before washing with TBS. Cells were then blocked for 45 min in 1% BSA, and following this, anti‐HA primary antibody (Biolegend, London, UK) was diluted 1:1000 and added for 1 h. Cells were then washed in TBS and blocked with 1% BSA for 15 min before addition of goat anti‐mouse IgG secondary antibody (Sigma‐Aldrich), diluted 1:1000, for 1 h. The resulting signal was detected using alkaline phosphatase incubation (Pierce PNPP substrate kit, Thermo Fisher, Swindon, UK), which bound to antibody‐tagged receptor; absorbance was read at 405 nm using a TECAN plate reader. Background absorbance was subtracted, and data are expressed as % cell surface loss of receptor.

The above protocol was used to fix, permeabilize, and block. Then, the cells preparations were incubated with anti-vimentin antibody, mouse monoclonal (clone V9, Sigma) at 1:1000 dilution in PBS with 10% NGS and 2% FBS, for one hour at RT. Followed by the incubation of goat anti-mouse IgG secondary antibody (Sigma) at a dilution 1:2000 for one hour at RT protected from light. Nuclei were stained with DAPI.

ELISA was used to assess anti-CSPG4 mAbs’ binding to CMPs and to inhibit anti-P10s serum antibodies’ binding back to the P10s peptide by VT68.2 mAb. For binding assays, ELISA plates (Immuno 4 HBX, Thermo Fisher Scientific) were coated overnight with 50 µL per well of 10ug/mL of peptides. After blocking (blocking buffer: PBS with 1% BSA), serially diluted antibodies were added to wells in blocking buffer and incubated at 37 °C for 2 h. Following washing at room temperature, goat anti-mouse IgG secondary antibody (Sigma-Aldrich, Inc., Saint Louis, MO, USA) was added in 1:15,000 dilution in blocking buffer for an hour’s incubation at 37 °C. The inhibition assay was performed similarly, except PADRE-conjugated P10s-immunized human serum [19 (link)] was added in a 1:800 dilution after washing off VT68.2 antibody and binding of the immunized serum antibodies to the peptide were visualized with an HRP-conjugated anti-human IgG (Sigma-Aldrich, Inc.), at a dilution of 1:10,000. Binding was detected by measuring absorbance at 450 nm using the Synergy LX multi-mode reader (BioTek©, Winooski, VT, USA).