Methods
Tissue collection and study population
11 placental biopsies were collected from Maastricht University Medical
Centre, the Netherlands, between November 2020 and May 2021. 3 placental
biopsies from uncomplicated pregnancies and 1 PE sample were collected
directly after birth. 6 Placental samples (n=2 PE) were collected from
the pathology department and received on formalin. Collection and usage
of the placentas was approved by the Medical Ethics Committee Academic
Hospital Maastricht and Maastricht University (METC, 16-4-047). Clinical
characteristics of all placental samples are summarized in Table S1.
Tissue dissection and fixation
Placental tissue (~20 cm3) was
sectioned from a central region of the chorion, stored for 24h at 4°C
and frequently washed with phosphate buffered saline (PBS) to remove
blood. Subsequently it was fixed (1h) in 4% paraformaldehyde (PFA) and
stored in PBS at 4°C.
Nuclear and vascular fluorescent dyes
Placental tissue slabs (~10mm3) were
fluorescently labelled to visualize nuclei and vascular structures.
Tissue was permeabilized in 0,1% triton, 1% BSA (20 min), and
incubated with 10µg/ml Ulex Europaeus Agglutinin I (UEA I), DyLight® 594
(Vector laboratories, CA, USA, cat#DL-1067-1,
emmax=618nm) for 1-3h, in the dark, at room temperature.
After washing with TBS, nuclei were labelled with 5µM SYTO 13 green
(Invitrogen, cat#S7575, emmax= 509nm) for 30 minutes in
the dark at room temperature.
Mounting and MPM imaging
For imaging, samples were mounted on a 50 mm glass bottom petri dish
(MatTek, Ashland, MA, USA) filled with PBS. MPM was performed with a
Leica TCS SP 5 (Leica Microsystems, Wetzlar, Germany) multiphoton
microscope. Excitation was at 780 nm. Fluorescence was collected with a
20x Leica HCX APO L, NA 1.0 water immersion objective or a 25x NA1.05
Olympus objective. Alternatively, the Leica Stellaris 8 Dive (Leica
Microsystems, Wetzlar, Germany) employing similar settings was used.
Autofluorescence and second harmonic generation (SHG) from unstained
samples was detected by an
external detector with bandpass filter 460-525 nm, and a forward
detector with 380-420 nm bandpass filter, correspondingly.
Signal of stained samples was detected using 4 channels. SHG was
collected as described before. Internal detectors with detection
bandwidth 444-496 nm, 508-559 nm, and 586-650 nm recorded
autofluorescence, nuclear stain, and vascular stain respectively.
All images were acquired in 8-bit, with bidirectional scanning mode and
at least 1024x1024 pixels. Z-step size ranged from 1.5 µm to 5µm. Image
stitching was performed with the
Leica Stellaris Dive microscope
equipped with motorized stage.