Fluromount g

A similar protocol was followed as with the immunoperoxidase stain; however H₂O₂ treatment was omitted. Chicken anti-GFP (Aves labs, Oregon, USA; 1:500) was added to either of the following, mouse anti-GFAP (1:500; Millipore), and mouse anti-NeuN (1:500; Millipore). The following day the sections were incubated with biotinylated secondary antibodies (goat anti-mouse) all at 1:1000 dilutions for 2 hours. Sections were incubated with a fluorescent secondary antibody for 2 hours; goat anti-chicken Alexa Fluor 488 (1:200; Invitrogen) and goat anti-mouse Alexa Fluor 568 (1:500; Invitrogen). The sections were treated with DAPI (5 mg/mL) in the dark for a couple of minutes. They were transferred to ice cold TBS. Each individual brain section was mounted on to slides and left to dry for a couple of hours. Once dried, a cover slip was added using Fluromount G (Ebioscience, Hatfield, UK).

Mouse placentas were fixed in paraformaldehyde overnight before being processed and embedded into paraffin wax for histological staining. Each sample was sectioned at 6 μm thickness perpendicular to the chorionic plate (Leica RM2235, Germany). Slides were then stained with hematoxylin & eosin (HE). All stained slides were covered by dibutyl phthalate polystyrene xylene mounting medium (Sigma) and scanned (Olympus VS120, Japan) to assess placental thickness and distinguish DJ and L layers. For immunofluorescence (IF) staining, the slides were incubated in heated antigen unmasking solution (Vector Laboratories, USA) for antigen retrieval, and the remaining steps were similar to those previously described (Dong et al. 2019 (link)). The primary antibodies for IF included Tpbpa (a marker for spongiotrophoblast cells, Abcam, #ab104401), Vimentin (a marker for interstitial cell, Abcam, #ab8978) and CD31 (a marker for endothelial cells, Abcam, #ab182981). The secondary antibodies included Goat anti-rabbit IgG Alexa Fluor 568 or 488 and Goat anti-mouse IgG Alexa Fluor 568 (Life Technologies). All IF slides were finally covered using Fluromount-G (eBioscience). Images were photographed and scanned using the Olympus VS120. We analyzed the intensity and density of positive regions on IF slides by Image J software (NIH, Bethesda, USA).

Post‐fixed brain hemispheres were sliced at the LEICA VT1000S vibrating microtome to obtain 50 µm thick sagittal sections. The brain sections were put into separate wells of a 24‐well plate and rinsed in a solution of 50 mM Tris‐Cl, 150 mM NaCl (TBS, pH 7.6) for 10 min. Permeabilization and blocking of non‐specific binding sites was achieved by incubation in 500 μl of blocking solution (10% [v/v] Normal Donkey Serum, Abcam; 1% [v/v] Triton‐X100, Sigma‐Aldrich; TBS) for 2 h at room temperature. The brain sections were then incubated in 500 μl of primary antibody solution overnight at 4°C. On the following day, the brain sections were rinsed six times in TBS. Each washing step was carried out for 10 min. The sections were then incubated for 2 h, at room temperature, in 500 μl of secondary antibody solution. Finally, the sections were rinsed six times with TBS and mounted on SuperFrost Plus Adhesion glass slides (Thermo Fisher Scientific) with Fluromount‐G without addition of DAPI (Thermo Fisher Scientific), unless otherwise specified. The glass slides were stored at 4°C, protected from light to prevent photobleaching.