Sections were incubated with primary antibodies for 30 min, after blocking endogenous peroxidase activity with 3% hydrogen peroxide, heating the slides in 10 m M sodium citrate in a microwave oven and blocking nonspecific protein binding in normal goat serum.
10 Internalised ranibizumab or bevacizumab were stained by incubation with AlexaFluor594-coupled polyclonal antibodies (1 10000) against human IgG for 90 min after blocking with 10% FCS/PBS for 30 min.
Control cells were pre-incubated with 1 50 dilution of unconjugated anti-PSGL-1 (cat. no. 556053; BD Pharmingen) for 60 min (after blocking) before being stained with PE-conjugated anti-PSGL-1 antibody.
Cells were fixed with 10% (wt/vol) formaldehyde for 20 min, and permeabilized with 0.5% Triton X-100 for 5 min. After blocking for 20 min at 37°C with 10% BSA, antigens were labeled with primary antibodies at 37°C for 1 hr.
The amount of prescribed analgesia used before and after the block was also noted and the staff was instructed not to give morphine within 60 min after the block being introduced.
The pain resolved completely, within 10 min after the block, but the patient had recurrence of headache after few hours.
In both groups, SBP, 5 min after spinal block, decreased from the baseline value.
Endogenous peroxidase was blocked with 3%H2O22 at room temperature for 10 min. After blocking for 45 min with 1.5% normal horse serum in PBS, the sections were incubated overnight at 4°C with primary Itgb3 antibody (1 200) in PBS.
The slides were incubated with hydrogen peroxide for 20 min following microwave heating with 10 mM citrate buffer (pH 6.0; Sigma-Aldrich, Germany) at 2-min intervals for a total of 10 min. After blocking with normal serum for 30 min, the slides were incubated with rabbit polyclonal antibody.
