Histamine

Figure 1C shows photographs
of the detection process. After preparing the HUVEC monolayers on
the membranes, a piece of the membrane was immersed in ECGM2 containing
5 mM [Fe(CN)₆]^4– on the device. The membrane
was pushed onto the device surface using a polydimethylsiloxane frame
(Dow Corning Toray, Japan). Then, Ag/AgCl (sat. KCl) reference and
Pt counter electrodes were inserted into the solution. A voltage of
0.5 V was applied to the 400 working electrodes and the current from
each electrode was obtained every 200 ms. The current values were
converted into colors to construct electrochemical images consisting
of 20 × 20 pixels.
For the assays using histamine, 45 μL
of 0.333 mM histamine (Merck, Germany) was added into ECGM2 containing
5 mM [Fe(CN)₆]^4– (final histamine concentration:
10 μM). The change in the current values was monitored in real
time..

Both flanks of adult anaesthetized mice were shaven. The next day, 100 µl of 1% Evans Blue (Sigma-Aldrich) in sterile saline (wt/vol) was injected intravenously through the lateral tail vein. In some experiments, mice received an intraperitoneal injection of pyrilamine maleate (4 mg/kg body weight in 0.9% saline; Sigma-Aldrich) before Evans Blue injection to inhibit release of endogenous histamine. 30 min after Evans Blue injection, 20 µl of PBS or PBS containing 50 ng VEGF164 (PeproTech), 300 ng SEMA3A (R&D Systems), or 50 ng histamine (Sigma-Aldrich) were injected intradermally each at three sites into the flank skin of anaesthetized mice. After 20 min, mice were culled, the skin was separated from the underlying muscle, and the tissue surrounding the injection sites excised (circled in Fig. 1 A) and dried overnight at 55°C. Evans Blue was extracted by incubation in formamide at 55°C overnight and quantified by spectrophotometry at 620 nm after subtraction of background absorbance at 740 nm. Data from the three sites injected with the same agent (ligand or PBS) were averaged and expressed as fold change relative to PBS control per each mouse. In some experiments, the inner side of the skin was imaged on an MZ16 stereomicroscope (Leica) equipped with a Micropublisher camera (Perkin-Elmer). In other experiments, a sample of liver or skin tissue was retained for immunoblotting.

Histamine and thrombin were purchased from Sigma-Aldrich (Munich, Germany). Histamine was used at 10 µM for the indicated times and thrombin was used at 10 U for 5 min. BAPTA-AM (Sigma-Aldrich, Germany) was preincubated for two hours at 5 µM. Diphenhydramine was used to inhibit activation of Histamine receptor type I, and ranitidine was used for inhibition of Histamine receptor type II. Both were preincubated at 1 µM and 10 µM, respectively, for 15 min. The ROCK inhibitor Y27632 (Sigma-Aldrich, Germany) was preincubated for 30 min at 10 µM.