Vaseline

The inner well is filled with 12.5 µl control medium consisting of MV3 medium without FBS. The prepared cover slip is then inverted and carefully lowered onto the chamber resting one edge before lowering gently to ensure the central chamber remains bubble free. Excess medium is blotted from the edges of the cover slip with care to avoid moving the cover slip, which could result in shearing of the cells over the bridge. The cover slip is then sealed into place with 1∶1 Vaseline:Paraffin (Melting point 58–62°C, Sigma-Aldrich). The outer surface of the cover slip is then washed with distilled water and dried before wiping with lens cleaning tissue. The chamber is then inverted to fill the outer chemoattractant well with approximately 80 µl MV3 medium using a 20 µl microloading tip (Eppendorf). FBS is therefore used as the chemoattractant as in other studies [26] (link). Holding the Insall chamber at 45 degrees whilst filling allows any bubbles to escape through the drilled holes. The two holes are then sealed with a thin strip of water resistant tape to avoid evaporation. The chemotactic responses of cells overlying the bridge region can then be analysed after incubating at 37°C for one hour to allow the gradient to form.

Ookinete cultures were added to an equal volume of Matrigel™ (BD) on ice, mixed thoroughly, dropped onto a slide, covered with a Vaseline-rimmed cover slip, and sealed with nail varnish. The Matrigel™ was then allowed to set at room temperature for at least 30 minutes. After identifying a field containing ookinetes, time-lapse videos (1 frame every 5 seconds, for 10 minutes) were taken of ookinetes using the differential interference contrast settings with a 63× objective lens on a Leica DMR fluorescence microscope and a Zeiss Axiocam HRc camera controlled by the Axiovision (Zeiss) software package. Speed of motility of individual ookinetes was measured by multiplying the number of body lengths moved by the length of the ookinete during the 10 minute video, divided by 10. Multiple independent slides and cultures were used for each parasite line. Video processing and annotations was carried out using the Axiovision or Axiovision LE (Zeiss) software.

Cells were plated on glass coverslips at very low density to obtain isolated single cells, incubated overnight in culture medium and used between 40 and 50 hs after plating. Coverslips were attached with Vaseline to the perforated bottom of 4–5 mL plastic chambers (35 mm cell culture dish caps) and placed on the stage of an upright microscope (BX50WI, Olympus) for patch-clamp work. Two discontinuous single-electrode voltage-clamp (DSEVC) amplifiers (SEC-05LX NPI, Germany) were used to simultaneously clamp the voltage and record membrane currents from two single cells in whole-cell voltage clamp (WCVC) mode, or from a single cell with one electrode in WCVC and the second in cell-attached voltage clamp (CAVC) mode. In some experiments, WC configuration was achieved but voltage clamp was not performed: instead, the resting membrane potential (Vm) was documented using the amplifiers’ bridge mode (BR) function. Cells were bathed in external solution (in mM: NaCl 140, KCl 4.7, CaCl₂ 1.8, MgCl₂ 1.2, Glucose 10, EGTA 0.1, HEPES 10) adjusted to pH = 7.2 and osmolarity 319–330 mOsm. Patch pipettes were filled with internal (in mM: KCl 138, MgCl₂ 3, TEACl 9, CaCl₂ 0.5, EGTA 9, Glucose 5, Na₂ ATP 5, HEPES 9; adjusted to pH = 7.4 and osmolarity 315–319 mOsm) or external solution.
Pulsing protocols were as follows: “steady HP”, continuously voltage clamping at any holding potential (HP); “−70 to +80 mV”, from an HP = −70 mV, 600 ms, +80 mV square pulses applied every 10–30 s; “±100 mV ramps”, from and HP = −50 mV, voltage ramps from −100 to +100 mV (slope ∼32 mV per sec) applied every 10–30 s; “step or IV”, from HP = −70 mV, 600 ms square pulses from −80 to +80 mV in 20 mV steps, every 5.5 s; “±80 paired pulses”, from HP = 0 mV, 10 s pulses to −80 and +80 mV, preceded by 100 ms prepulses at ±10 mV, every 30 s; “long pulses”, HP at any given value, usually −80, +80 mV or 0 mV, held for variable periods >1 s. Recordings were acquired at 13 kHz and low pass filtered at 1 kHz. Channel recordings were further filtered (100–200 Hz) and decimated (10–50) for ease of display. Macroscopic Im was analyzed with Clampfit10 (Molecular Probes) and Excel (Microsoft). For single channel event amplitude, long traces were surveyed with the Histogram function of Clampfit10 to locate sections with discrete transitions, then transferred those sections to Excel to produce refined all-points histograms with bin sizes of 0.25–0.5 pS; traces and histograms were plotted with SigmaPlot (SPSS Inc.). When suitable, results are reported as average ±SEM.

A tongue-shaped flap was created on the radial wall of the 5th digit, with the longitudinal edges not exceeding the radial surface of the digit, and the distal edge made slightly beyond the PIP joint line. To ensure full coverage of the volar skin defect, the flap was made 2 mm larger in diameter than the recipient site (Fig. 2).Figure 2

Representative illustrations of camptodactyly of the 5th digit. (a) Frontal and lateral views of the 5th digit before surgery. (b) Design of the tongue-shaped flap, with the longitudinal edges limited within the radial surface, and the distal edge made slightly exceeding the proximal interphalangeal joint line. (c) The volar incision. (d) The lateral view of the digit flap transfer, with direct suturing performed for closure of the donor site. (e) The volar view of the digit after flap transfer, with complete coverage of the volar skin defect.

While creating the edges of the flap, care was taken to preserve the perforating blood vessels of the proper palmar digital arteries, as well as the proper palmar digital nerves.
Sequential release of affected soft tissues was performed in the following order—skin, subcutaneous fibrous fascia, flexor digitorum superficialis tendon, lumbrical muscle insertions if present, and volar plate. The degree of passive extension of the PIP joint was repeatedly tested, and surgical release was considered complete upon achieving full passive extension of the joint. Kirschner (K)-wire fixation was performed following volar plate release.
The radial flap was rotated 90° to cover the volar skin defect, and direct suturing was performed to close the donor site. Free skin grafting was indicated in the presence of high suture tension.
Mupirocin ointment and petroleum jelly (Vaseline) were subsequently applied, and the wound was wrapped with clean dressing. All digits were immobilized in the extended position with a cast for three weeks.