In relation to a classical bulk thermolysis (390°C, 6 h) which affords branched waxy polymers, dilution with phenylether permits controlled diffusion reactions modifying the product characteristics: low molar masses, narrow distribution, high crystallinity, rich in α-olefins and few long branching formation.
After washing with phosphate-buffered saline (PBS), they were incubated with secondary antibodies against rabbit IgG conjugated to a peroxidase-labeled dextran polymer (no dilution, anti-rabbit Envision, Dako Japan, Tokyo) or against mouse IgG conjugated to a peroxidase-labeled dextran polymer (no dilution, anti-mouse Envision, Dako Japan) for 1 hour.
After the reaction with goat anti-mouse IgG or goat anti-rabbit IgG conjugated to peroxidase-labeled dextran polymer (no dilution; En Vison+ Mouse or En Vison+ Rabbit; DakoCytomation Norden A/S), the color was developed with 3,3'-diaminobenzidine tetrahydrochloride in 50 mM Tris-HCl buffer, pH 7.6, containing 0.006% H2O2.
This RBC suspension (90 μL) was then mixed with each of the polymer serial dilutions (10 μL) on a sterile 96-well round-bottomed polypropylene microplate (RBC concentration on plate is 1.0 × 10 RBC/mL).
Due to low CMC, the polymeric micelles remain stable at low polymer concentration after dilution with body fluids.
The intensity of the B band, which was tentatively ascribed to the emission of polymer, increases with the dilution.
The data were compared with the thermodynamic functions of test solutes in polymer solution at infinite dilution.
Immunostaining was performed using the Vision Biosystems Bond Immunostaining Robot (Leica Microsystems, Wezlar, Germany) under normal operating conditions as described in our previous study [21] using a sheep-anti-BrdU antibody (1∶6000; Fitzgerald, UK) or Hypoxyprobe mouse monoclonal antibody (Vision Biosystems mouse polymer protocol at 1∶50 dilution).
After incubation in blocking solution (Zytomed ZytoChem-Plus AP Polymer Kit, Berlin, Germany), 1 : 50 dilution of anti-MGMT clone MT23.2 (Zymed Laboratories, Amsterdam, NL , USA was added and the slides were incubated overnight at 4°C.
To verify whether identified antibodies were interfering with the assay itself, a competitive ELISA was performed in which the plate was coated with polymer-specific mAb2C1, loaded with excess polymer antigen, and different dilutions of cell culture supernatants were applied.
