Ab52455

Placental tissues of normal patients and those of patients with preeclampsia and intrauterine growth restriction were obtained in their third trimester (32 weeks of gestation; n = 3) during cesarean sections. The use of the tissues was approved by the Clinical Research Ethics Committee of Kyushu University and Tokyo University of Pharmacy and Life Sciences (#1512), and informed consent was obtained from the participants. The tissues were directly fixed in 0.2 M phosphate buffer (pH 7.45, room temperature) containing 4% paraformaldehyde for 8 h before being embedded in paraffin and sectioned into 6-μm sections. Before the tissues were immunostained for NYNRIN or major histocompatibility complex class I, G (HLA-G), the sections had been deparaffinized, rehydrated, and boiled in 10 mM citrate buffer (pH 6.0) for 20 min. The sections were then incubated overnight at 4 °C with a rabbit polyclonal anti-NYNRIN antibody (HPA018945; 1:100; Atlas Antibodies, Stockholm, Sweden), a mouse monoclonal anti-HLA-G antibody (ab52455; 1:100; Abcam, Tokyo, Japan), or a normal rabbit IgG (sc-2027; 1:100; Santa Cruz Biotechnology, Dallas, TX, USA). The sections were subsequently incubated with Histofine Simple Stain MAX-PO (Nichirei Biosciences) and DAB (Fujifilm Wako Pure Chemical Corp.), counterstained with hematoxylin, and examined under the digital microscope.

PFA-fixed coronal brain sections (10 ​μm) were thaw-mounted onto Superfrost™ plus slides (Thermo Scientific, USA) and immunofluorescently labeled with HLA-G to identify the presence of hAECs in the brain following TBI. Sections were fixed in 4% PFA for 5 ​min, washed in 0.01 ​M PBS (2 ​× ​5 ​min), and then blocked with a Mouse on Mouse (M.O.M.™) Ig blocking reagent (Vector Laboratories, USA) for 1 ​h. Following wash with 0.01 ​M PBS (2 ​× ​2 ​min), sections were incubated in M.O.M.™ diluent (Vector Laboratories, USA) for 5 ​min, prior to HLA-G antibody incubation for 30 ​min (1:500; Ab52455; Abcam, UK). Sections were then washed with 0.01 ​M PBS (2 ​× ​2 ​min) and incubated with M.O.M Biotinylated Anti-Mouse IgG reagent (Vector Laboratories, USA) for 10 ​min. Following washes with 0.01 ​M PBS (2 ​× ​2 ​min), fluorescein-Avidin DCS (Vector Laboratories, USA) was applied for 5 ​min. Sections were then washed with 0.01 ​M PBS (2 ​× ​5 ​min), cover-slipped with Vectashield DAPI® (Vector Laboratories, USA), and examined with an Olympus fluorescence microscope. Positive HLA-G stains were confirmed upon co-localization with DAPI⁺ nuclei.