Goat anti rat igg hrp

To obtain cell lysates, hMSCs were suspended in 150mM NaCl, 50mM Tris-HCl (ph 7.4), 0.02% NaN₃, 20 µg/mL PMSF and protease inhibitor cocktail (Roche), sonicated, and then solubilized in 2% Nonidet P-40 (NP-40). Protein samples were then separated on a 7.5 % SDS–PAGE electrophoresis gel (Bio-Rad). Resolved membrane proteins were transferred to Polyscreen polyvinylidene difluoride (PVDF) membranes (Bio-Rad) and blocked with 10% nonfat dry milk and 0.1% Tween20 in TBS. For assessment of E-selectin ligands, membranes were incubated with recombinant mouse E-selectin–human Ig chimera in TBS 0.1% Tween20 containing 2mM CaCl₂, followed by staining with rat anti-mouse CD62E mAb (R&D Systems) and then goat anti-rat IgG-HRP (Southern Biotech). For detection of sLe^X, membranes were incubated with rat HECA452 mAb, followed by staining with goat anti-rat IgM-HRP (Southern Biotech). For detection of CD44, membranes were incubated with mouse anti-human CD44 (Clone 2C5; IgG₂), followed by staining with goat anti-mouse Ig-HRP (Southern Biotech). HRP conjugated antibodies were detected by chemiluminiscence using Lumi-Light Western blotting substrate (Roche).

Rats were anesthetized with an isoflurane/oxygen vapor mixture (isoflurane 5% induction, 1–3% maintenance), and blood was collected from the jugular vein during Weeks 1–6, and 10. Antibody titer was defined by the dilution required to achieve a 50% signal using enzyme-linked immunosorbent assay (ELISA) with a Biomek 4000 liquid handling robot. 96-well assay plates were coated with 25 µg/well cathinone-BSA conjugate and blocked with skim milk. Twelve 1:1 rat plasma dilutions were added to the plate starting at 1:200 and allowed to incubate for 2 h. Following a wash step, goat anti-rat HRP IgG (SouthernBiotech) at 1:10,000 dilution was incubated in the plates for 18 h at 4 °C. After a second wash step the plates were developed using a 3,3’–5,5’-tetramethylbenzidine (TMB) substrate kit (Thermo Pierce) and 2 M H₂SO₄ as a stopping solution. The well absorbance values were read at 450 nm and normalized to the highest value for each sample in GraphPad Prism version 6, followed by curve fitting with log(inhibitor) vs. normalized response – variable slope to find the midpoint titer. Competitive ELISA was also performed in a similar manner but with an added step: plasma at the IC₈₀ dilution was incubated with free drug (α-PVP, MDPV or methamphetamine) dilutions of 1 mM to 0.1 nM (eleven 5-fold dilutions) in cathinone-BSA coated plates for 2 h.

For characterization of anti-MA antibody titers, rats were anesthetized with an isoflurane/oxygen vapor mixture (isoflurane 5% induction, 1–3% maintenance), and blood was collected from the jugular vein during weeks 1–6, 14, 20 and 30. Antibody titer was defined by the dilution required to achieve a 50% signal using enzyme-linked immunosorbent assay (ELISA) with a Biomek 4000 liquid handling robot. 96-well assay plates were coated with 25 μg/well MH6-BSA conjugate and blocked with skim milk. Twelve 1:1 rat plasma dilutions were added to the plate starting at 1:200 and allowed to incubate for 2 h. Following a wash step, goat anti-rat HRP IgG (SouthernBiotech) at 1:10,000 dilution was incubated in the plates for 18 h at 4 °C. After a second wash step the plates were developed using a 3,3′-5,5′-tetramethylbenzidine (TMB) substrate kit (Thermo Pierce) and 2 M H₂SO₄ as a stopping solution. The well absorbance values were read at 450 nm and normalized to the highest value for each sample in GraphPad Prism version 6, followed by curve fitting with log(inhibitor) vs. normalized response – variable slope to find the midpoint titer. One individual sample was unavailable in Week 6 and one animal was lost to the study prior to the Week 14, 20 and 30 time-points; the values were replaced with the group average for the time point for statistical analysis.