S3022

The 5 μm-thick frozen mouse sections were air-dried for 20 min at RT, acetone fixed (5 min, RT), and air-dried. After peroxidase quenching with 1% aqueous H₂O₂ in methanol for 15 min and a rinse in tris-buffered saline with Tween (TBST), sections were blocked in protein block solution (Dako #X0909) with 10% donkey serum (30 min, RT), then incubated with the primary rabbit antibody to mouse PD-L1 (Genentech #GEN130-47-4; diluted to 10 μg/mL in antibody diluent (Dako #S3022)) in a humidity chamber at 4 °C overnight. Then, a biotinylated donkey anti-rabbit IgG secondary antibody (Jackson Immunoresearch #711-065-152; 1:200 in antibody diluent) was applied at RT for 30 min. Detection involved an ABC-HRP kit (VECTASTAIN Elite # PK-6100), a metal enhanced DAB substrate kit (Thermo Scientific #34065), and haematoxylin counterstaining. Bright-field images were taken with a Leica DM LB2 microscope and DFC 300 FX camera.

Whole mouse brains were perfused with an ice-cold PBS and fixed in 4% neutral-buffered formalin. A fixed brain was embedded in an optimal cutting temperature compound (OCT), and cryosection (20 μm) was performed by cryostat microtome. Immunofluorescence staining was performed according to the following procedure: frozen tissue slices were removed from −20 °C and placed into room temperature for 10–20 min. Slices were further rehydrated and washed by PBS for 10 min. Tissue non-specific binding was performed by adding the blocking buffer for 1 h (PBS containing 5% bovine serum albumin). After 1 h, the tissue slices were washed twice and stained with primary antibodies against active caspase-3, the neuron marker NeuN, and the activated macrophage marker Iba-1, overnight in 4 °C. The primary antibody was diluted in antibody diluent (Dako S3022), and antibody concentration was determined according to product sheet suggestion. After one night, slices were washed twice and stained with secondary antibody for 2 h. DAPI was used for nuclear staining for 30 min. Fluorescent tissue sections were visualized under a fluorescence microscope (Ax10, Zeiss, Oberkochen, Germany).

For analysis of the localization of vWF, α-SMA, active β-catenin, and FPR2 in vivo or in vitro, primary antibodies against vWF (1:200, Abcam, Cambridge, MA, USA, ab6994), α-SMA (1:200; Dako, M0851), active β-catenin (1:100; Cell Signaling Technology, Danvers, MA, USA, 8814S), and FPR2 (1:100; Novus, St. Louis, MO, USA, NLS1878) was added to antibody diluent (Dako, S3022) and reacted with slides at 4 °C overnight. The secondary antibody Alexa Fluor 488 and 594 (1:400; Invitrogen, Camarillo, CA, USA, A21206 and A11012) was added for 1 h. The slides were counterstained with 4,6-diamidino-2-phenylindole (DAPI; Invitrogen, D3571). The images were observed with a confocal microscope (LSM 700; Zeiss, Oberkochen, Germany). The observed images were analyzed with ZEN blue software (Zeiss). The experiments were performed in triplicate.

TWOD consists of red ginseng, Astragalus, Panax, leech, earthworm, Salvia, and Poria (weight ratio 2 : 15 : 2 : 3 : 2 : 10 : 15). All ingredients of the TWOD were combined and boiled for 2 hours. After boiling, TWOD was filtered and concentrated to 1 g/ml. Then through sterilization and vacuum packaging, TWOD was stored at 4°C at the China-Japan Friendship Hospital Extracting Room.
CaD was purchased from Xi'an Lijun Pharmaceutical Co. Ltd. Anti-occludin (ab31721), anti-claudin-5 (ab53765), anti-MMP-9 (ab137867), and horseradish peroxidases (HRP, ab97069) were purchased from Abcam. The antibody diluent (s3022) was obtained from DAKO. Trypsin (No. 0458) was get from Amresco. STZ (s0130) was purchased from Sigma.
The TWOD group was treated orally with TWOD, which was diluted in 5 ml of distilled water at a daily dose of 10 g/kg and considered as a therapeutic agent for the prevention of DR. The CaD group was used as the positive control group and was treated orally with CaD at the daily dose of 250 mg/kg diluted with 5 ml of distilled water. The control group and model group were treated with 5 ml distilled water over the same treatment period. The rats receive TWOD and CaD for 9 months.

Samples were fixed in 4% paraformaldehyde (24 h, 4 °C), dehydrated, and embedded in paraffin, according to standard histological protocols. Sections (5 μm) of tissues were mounted on SuperFrost Plus slides (Thermo Fisher, Waltham, MA, USA). After dewaxing and rehydratation, sections were heated at 97 °C in 10 mM citrate buffer (pH 6.0) for 40 min. Nonspecific binding was blocked using protein block serum-free (X0909; DakoCytomation) for 1 h at room temperature. Sections were incubated with primary antibodies diluted in antibody diluent (S3022; DakoCytomation) overnight at 4 °C. The primary antibodies used were anti-Z0–1 (1:500; LifeTech), anti-claudin-1 (1:500; InVitrogen), anti-claudin-2 (1:500; InVitrogen), anti-PCNA (1:1000; GeneTex) and anti-Ki67 (1:50, DakoCytomation). Nuclei were stained with Hoechst before mounting the slides using Fluorescent Mounting Media (Dako). Sections were scanned using a Pannoramic scan digital slide scanner (3DHistech) and analysed using digital slide scanner Pannoramic scan (3Dhistech). For Ki67 and PCNA 10 crypts were measured per mice and per cut.

Human cadaveric IVD samples (Table 1) of AF (N = 6), NP (N = 9) and CEP (N = 7) were assessed via IHC using SAM11, a primary antibody for PAR2 (1:50; Invitrogen 35-2300). Briefly, tissue slides were deparaffinized, rehydrated, blocked for endogenous peroxidase activity (0.3% H₂O₂ in MeOH), and antigens retrieved using a citrate buffer (90°C, pH 6.0) for 20 min. Blocking for non-specific binding was performed with 5% goat-serum (1% BSA-PBS, 5% goat serum, 0.05% Tween, and 0.05% sodium azide). Primary antibodies were incubated for 2.5 h in background reducing antibody diluent (Dako S3022) followed by incubation with secondary antibody: biotinylated goat anti-mouse (1:200, VectorLabs BA9200). Finally, tissue sections were incubated with streptavidin-horse radish peroxidase and developed with 3,3-diaminobenzidine (DAB) for 90 s (VectorLabs SK-4100). Tissue was counterstained with Gills No. 2 Hematoxylin (Electron Microscopy Sciences 26030-20) and slides dehydrated and mounted. Normal human lung tissue was used for positive and negative controls, with omission of primary antibody for the latter. All antibody concentrations and DAB times were kept consistent across experimental and control samples. Each tissue sample was quantified as described prior (Wiet et al., 2017 (link)).