Fw4000

Twenty islets maintained in cell RPMI supplemented with 10 % SVF were treated during 6 hrs by 100 μM H₂O_2, then washed by centrifugation (500 g, 5 min.) and incubated with fluorescein diacetate (0.67 μM) and propidium iodide (4 μM) before observation by fluorescent microscopy (Leica FW 4000, ×20 objective). Nine random fields were analysed per sample. Fluorescence intensity was analysed using the ImageJ software. Results are expressed as green intensity/islet surface unit. Data were obtained from four different islet preparations.

SMP (30‐60 nmol/L) was stained by 2 μmol/L of the red fluorescent PKH26 lipid probe (Sigma). PKH26‐stained SMPs were washed twice by centrifugation (14 000 g, 60 minutes) in HBSS at 4°C before incubation with P1ECs during 6‐48 hours. Capture of PKH26‐stained SMPs by target ECs was assessed after three washings by fluorescent microscopy (Leica FW 4000) and quantified by flow cytometry. The efficacy of the SMP capture was expressed as the percentage of red fluorescent ECs (Guava Easy Cyte Plus System, Millipore).

Video fluorescence microscopy measurements were carried out using a Leica FW4000 and 40× objective (Platform of Quantitative Imagery, Faculté de Pharmacie, Université de Strasbourg). Acquisition of fluorescence images was carried out at an interval of every 5 s using the software MetaMorph (Molecular Devices). The experiment was divided into two acquisition periods; an initial acquisition of 10 min where cells were incubated in NES/YO-PRO-1 solution where YO-PRO-1 is at a concentration of 10 μM (iodide salt, Thermo Fischer). The solution was then gently exchanged for a NES/YO-PRO-1 solution containing BzATP and inhibitors (or BzATP alone) for a second acquisition period of 15 min. This second solution containing inhibitors was prepared immediately prior to application, and concentrations used are the same as those used for electrophysiology. Cells were maintained at 37 °C during measurements.
Confocal imaging was captured with Leica SPE using oil immersion objective: 63×, Numerical Aperture 1.4. Excitation (λexc) and emission (λem) wavelengths were as follows: Hoechst (λexc = 364 nm; λem = 430–481 nm), Alexa 488 (λexc = 495 nm; λem = 500–600 nm) and mScarlet (λexc = 561 nm; λem = 648–708 nm).

With the rats (Group C, n = 10) lying supine under anesthesia, a median incision of the hypogastrium was made to expose the anal canal. CB-HRP (10 to 15 μl, 3 μg/μl) (Sigma, St Louise, MO, USA) was slowly injected into the anal canal wall using a micro-syringe into three sites equally distant from one to another. The needle was retained for 15 min and medical adhesive was applied to seal off the needle holes. The rats were kept alive for 48 h. Then, the rats were perfused transcardially with phosphate buffered saline (PBS), followed by buffered 4% paraformaldehyde. Spinal cords and bilateral dorsal root ganglia from T12 to S4 were removed and placed in PBS containing 20% sucrose at 4 °C for 24 hours. Forty-μm sections were obtained using a cryostat-microtome. HRP-positive neurons were shown using tetramethylbenzidine-sodium tungstate (TMB-ST) and observed using an Olympus BH-2 microscope equipped with brightfield and darkfield optics (Olympus, Tokyo, Japan). The proportions of CB-HRP-positive neurons in different segmental dorsal root ganglia were calculated using the Leica FW4000 image analysis system using one slide per animal.
CB-HRP positive neurons and afferent fibers of rat rectum (Group D, n = 10) from different spinal segments were analyzed using the same methodology as for the anal canal.