Parafilm

For retrograde labeling of trigeminal sensory ganglion neurons innervating the dura mater, rats (n = 2) were anaesthetized with a combination of ketamine (Calypsol, 70 mg/kg, i.p., Gedeon Richter, Hungary) and xylazine (CP-Xylazin 2%, 10 mg/kg, i.p., Produlab Pharma, Netherlands). The head of the animal was stabilized in a stereotaxic frame, the scalp was incised in the midline and the parietal bone was exposed on one side. A cranial window was drilled into the parietal bone to expose the underlying dura mater. The fluorescent dye True blue (Sigma-Aldrich, Germany) was used for retrograde tracing. 10 μl of True blue (2% dissolved in SIF) was applied onto the exposed surface of the dura mater. After 5 min, the application site was covered with a piece of parafilm (Merck, Germany) and the overlying skin was closed by a suture. All surgical procedures were performed under aseptic conditions. Postoperatively the animals received diclofenac potassium (Cataflam 15 mg/ml, Novartis, Switzerland) offered in the drinking water (10 mg/kg body weight). After a survival period of 5 days, the animals were perfused transcardially and the trigeminal ganglia were removed and processed for immunohistochemistry.

Following preconditioning, the plates were opened and air-dried under a laminar flow cabinet. For Striga, a new labeled plastic petri plate was supplemented with a 90 mm Whatman filter paper ring, supplied with 900-µL sterilized milliQ water, and six dried disks containing preconditioned seeds. A 55-µL of germination stimulant was added on each disk. For Phelipanche/Orobanche, six dried disks containing preconditioned seeds were transferred into a 24-well plate and 100 µL of germination stimulant was added on each disk. Plates were sealed with parafilm (Merck KGaA, Darmstadt, Germany) and incubated at 30°C for Striga (24 h) or 25°C for Phelipanche/Orobanche (5 d). A protocol describing the critical steps is depicted in Supplemental Figure S8.

Liverpool (LVP) strain Aedes aegypti [24 (link)] were used for outcrossing and as the injection strain. LVP and transgenic lines were maintained at 28°C, 70% relative humidity on a 12/12 hour light/dark cycle with 1 hour of dawn and dusk, and provided with 10% sucrose solution ad libitum. Larvae were vacuum hatched in water containing Interpet Liquifry no. 1 (Interpet, Surrey, UK) and then reared in pans and fed with ground TetraMin flakes (Tetra, Herrenteich, Germany). Cages of adult mosquitoes were blood fed with defibrinated horse blood (TCS Bioscience) using a Hemotek membrane feeding system (Hemotek), with the reservoir covered with parafilm (Merck). Eggs were collected onto wet coffee filter paper and stored dry until hatched.

Approximately 1 cm² of a wound dressing was added to a polypropylene vial containing 100 µL of SAAP-148 in PBS (76 nmol). After 1 h incubation at 37 °C and 5% CO₂, the wound dressing was removed and the remaining volume of SAAP-148 containing solution was determined using a pipette. The following wound dressings were tested: gauze (BSN Medical GmbH, Hamburg, Germany), Mepilex border (Mölnlycke Health Care AB, Gothenburg, Sweden), Opsite Post-Op (Smith & Nephew Medical Limited, Hull, UK), Cuticell (BSN Medical GmbH) and Tegaderm film (3 M Health Care, Neuss, Germany). The recovered SAAP-148 containing solutions after incubation with a wound dressing were compared to the same solution without a wound dressing or with a low-adhesive material Parafilm (Merck, KGaA, Darmstadt, Germany), which was used in the in vivo study of de Breij et al. [12 (link)].

Parasitic seeds require specific temperature and moisture conditions for a certain period of time (preconditioning) to germinate. For this purpose, Striga seeds were surface-sterilized in a 50-mL tube with 20% bleach with 0.1% Tween-20 for 5 min. For Phelipanche/Orobanche, seeds were surface-sterilized in a 50-mL tube with 75% ethanol for 3 min. After removing the bleach/ethanol with subsequent six washings, the seeds were again sterilized with 3% bleach for 3 min, poured along with bleach, were poured in vacuum assembly (Sigma Aldrich) placed under a laminar flow cabinet, and thoroughly rinsed with six subsequent washings with sterilized milliQ water. The seeds were shifted in a glass petri plate and air-dried under a laminar flow cabinet for 3–4 h. Approximately 9-mm glass fiber disks were put in a glass petri plate and parasitic seeds (∼50–100) were spread uniformly on each disk. Then 12 disks were carefully transferred on a sterilized 90 mm Whatman filter paper (Merck KGaA, Darmstadt, Germany), placed in a plastic petri plate and moistened with 3-mL sterilized milliQ water. The plates were sealed with parafilm (Merck KGaA, Darmstadt, Germany) and covered in an aluminum foil. The plates with Striga seeds were put in an incubator at 30°C for 10 d, while the ones containing Phelipanche/Orobanche seeds were incubated at 21°C for 14 d.

T2-weighted MRI was performed on day 1 to delineate the infarct. After anesthesia (5% isoflurane/air, Forene; Abbott), the mice were positioned on a heated MRI cradle and fixed by bite and ear bars. The animals were continuously supplied with 2% isoflurane/air until the end of the experiment. Respiration and body temperature (37.0°C ± 0.5°C) were constantly monitored. A small sheet prepared from 1% v/v agar/water solution was placed directly on the animal’s head to reduce susceptibility artifacts and covered with parafilm (Merck KGaA). Two different systems were used.
With the first system, the cradle was manually positioned in the center of the 9.4-T small-animal MRI scanner (Biospec 94/20; Bruker Biospin GmbH). All images were processed and generated using Paravision 5.1 (Bruker Biospin MRI). T2-weighted images were acquired with a fast spin-echo sequence (rapid acquisition with relaxation enhancement) (repetition time, 7,700 ms; effective echo time, 100 ms; rapid-acquisition-with-relaxation-enhancement factor, 30; field of view, 2 × 2 cm; slice thickness, 0.5 mm; interslice distance, 0 cm; number of slices, 20; matrix, 192 × 192; number of averages, 8).
With the second system, a T2-weighted fast spin-echo 2-dimensional sequence was acquired in a 1-T nanoScan PET/MRI scanner equipped with an MH20 coil (resolution, 0.27 × 0.27 × 0.9 mm; Mediso Medical Imaging Systems).

Each isolated species was cultured to 0.01 (OD 580 nm) as per the planktonic growth curves. Aliquots of 100 µl of each dilution for each species was placed into a 96 well plate and a Nunc Immuno TSP 96 peg lid (Fischer Scientific, 445497) was applied. The plates were sealed with parafilm (Sigma P7793) and incubated at 25 °C for 72 h to allow all of the species sufficient time to produce a mature biofilm⁷⁵ .
The mature biofilm was quantified by staining with crystal violet for 2 min followed by two ten min washes in an excess of deionized 18MΩ water. Crystal violet which was bound to the biofilm matrix was eluted in 100% ethanol and the eluate read on a BMG Fluostar Optima at 570 nm. All results were produced from the mean of three repeats.