Zo1 1a12

Organoids and marmoset’s corneas were fixed in 4% paraformaldehyde for 4 h at 4 °C followed by washing in PBS three times for 15 min. Organoids and marmoset’s corneas were allowed to sink in 30% sucrose overnight and then embedded in OCT and cryosectioned at 12 µm. Marmoset’s corneas were vertical embedded and sectioned. Sections were permeabilized in 0.2% Triton X-100 in PBS and blocked in block buffer (2% BSA 5% fetal bovine serum) for 1 h at room temperature. Sections were subsequently incubated with the indicated primary antibodies at a 1:100 dilution in block buffer at 4 °C overnight. Secondary antibodies used were donkey Alexa Fluor 488, 568 and 647 conjugates (Invitrogen, 1:1000). After staining with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) in PBS for 5 min, slides were mounted in Vectashield anti-fade reagent (Vector Laboratories). Confocal imaging was performed with Leica TCS SP8 DLS LightSheet microscope. Primary antibodies: PAX6 (rabbit, ab5790), CHX10 (rabbit, ab133636), ZO-1 (mouse, ThermoFisher ZO1-1A12), MAP2 (chicken, ab5392), S100β (rabbit, ab52642), RAX (rabbit, ab23340), CD31 (mouse, ab23340), aSMA (rabbit, ab5694), DAPI (49,6-diamidino-2-phenylindole), ThermoFisher D1306, NeuN (mouse, Sigma-Aldrich MAB377), AAV (rabbit ab45482), Cas9 (Sp)(E7M1H) XP^® (rabbit, CST #19526, CK3 (mouse, ab68260).

The cells were washed with PBS and fixed in 4% paraformaldehyde (PFA) at room temperature (RT) for 30 minutes. The cells were permeabilized with 0.5% Triton X-100 in PBS for 30 minutes. After blocking with 2% goat serum for 2 hours at RT, the tissues were incubated overnight at 4 °C with primary antibodies [anti-ZO-1, 1:200 (ZO1-1A12, Thermo Fisher Scientific, Rochester, NY, USA); anti-Ki-67, 1:200 (MIB-1, Milan, Italy); anti-vinculin, 1:200 (Abcam, Cambridge, Massachusetts, USA). The samples were incubated with goat anti-mouse fluorescein isothiocyanate (FITC)-conjugated secondary antibody in 20% goat serum for 2 hours at RT. After each step, the cells were washed 3 times with 1X PBS. After removing the wall of the Lab-Tek slide, the cells were covered with mounting medium and cover slips. Cells were examined with an LSM 510-meta laser scanning microscope (Zeiss, Milan, Italy). Cells were examined under ultraviolet light or by excitation at wavelengths of either 488 nm or 594 nm where subsequent detection of the fluorescence was obtained.

Cells were fixed by 4% PFA treatment for 15 min and stored in PBS at 4 °C. Blocking was performed for 30–45 min in blocking buffer: 1% BSA and 0.5% Triton X for nuclear staining or 0.1% Tween 20 for surface or intracellular staining and 0.1 M glycine in PBS. Following three PBS washes, primary antibody was added in blocking buffer without glycine and incubated overnight at 4 °C. The primary antibodies used are as follows: Anti-WT1 1:200 (Abcam; ab89901), Anti-ZO1 1:100–300 (Invitrogen; ZO1-1A12), Anti-POSTN 1:200 (ThermoFisher; PA5-98301), Anti-α-SMA 1:200 (Abcam; ab7817) and Anti-Ki67 1:200 (BioLegend; 350502). The next day, the cells were washed thrice with PBS, secondary antibody (Invitrogen, 1:1000–2000) was added in 1% BSA-PBS and incubated for 2 h at room temperature. The secondary antibodies were used are as follows: goat-anti-mouse-Alexa488 (A11001), goat-anti-rabbit-Alexa546 (A11010), goat-anti-mouse-Alexa546 (A11030), goat-anti-mouse-Alexa594 (A11032), goat-anti-rabbit-Alexa647 (A21245), goat-anti-mouse-Alexa647 (A21236). Following three washes with PBS, Hoechst or DAPI (5 ng/ml, 1:10,000) was added for nuclear counterstaining.

The following primary antibodies were used: rabbit monoclonal MARVELD2 antibody (IF: 1:100; 54H19L38, Thermo Fisher Scientific), mouse monoclonal ZO‐1 antibody, Alexa Fluor 488 or 647 (IF: 1:100; ZO1‐1A12, Thermo Fisher Scientific), mouse monoclonal E‐cadherin antibody (IF: 1:100, WB: 1:1000; 610181, BD Biosciences), mouse monoclonal N‐cadherin antibody (IF: 1:250, WB: 1:2500; 561553, BD Biosciences), rabbit monoclonal phospho‐β‐catenin (Ser552) (IF:1:100, WB: 1:1000; 9566, Cell signaling).
Secondary antibodies were chicken anti‐rabbit Alexa 647 (1:400, Thermo Fisher Scientific, A‐21443), donkey anti‐mouse Alexa 555 (1:400, Thermo Fisher Scientific, A‐32773), anti‐rabbit IgG HRP‐linked (1:2000, Cell Signaling, 7074), anti‐mouse IgG HRP‐linked (1:2000, Cell Signaling, 7076).

Western blot analysis was performed for tight junction proteins (zonula occludens-1 (ZO-1), Occludin and Claudin-5) and glial fibrillary acidic protein (GFAP) from amygdala brain tissue homogenates. Lysates were made using RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0), centrifuged at 16,100 × G for 30-min and total protein levels were estimated from supernatant using a BCA kit (23225, Thermofisher). Western blot samples were made using XT sample buffer (1610791, Biorad) with DTT and boiled at 95℃ for 10 min. Samples were resolved in duplicate 4–12% BIS–TRIS gels (3,450,125, Biorad) under reducing condition and transferred to PVDF membrane. Probing was done against ZO-1 (1:1000; ZO1-1A12, Thermofisher), Occludin (1:1000; OC-3F10, Thermofisher), Claudin-5 (4C3C2, Thermofisher), GFAP (1:1000; G3893, Sigma) and beta-actin (1:5000; 8H10D10, Cell Signaling). Signals were detected using chemiluminescence substrate (34075, Thermofisher) with anti-mouse IgG-HRP (1:10,000; GENA93, Millipore Sigma), anti-rabbit IgG-HRP (1:20,000; GENA934, Millipore Sigma) or with fluorescence signals using IRDye 68RD goat anti-mouse (1: 10,000; 926–68070, Li-Cor) and IRDye 800 CW goat anti-rabbit (1:10,000; 926–32211, Li-Cor). Protein levels were quantified by densitometric analysis using ImageJ software.

The cultured cells were washed with PBS and fixed in 4% paraformaldehyde (PFA) at RT for 20 minutes. The cells were permeabilized with 0.5% Triton X-100 in PBS for 30 minutes (note: the permeabilization step was not performed for 2A12 considering that it is a cell surface marker and it may damage the epitope). After blocking with 10% bovine serum albumin (BSA) for 2 hours at RT, the cells were incubated overnight at 4°C with primary antibodies such as anti-Ki-67 (n = 4) (1 : 200 (MIB-1, Milan, Italy)) and anti-2A12 (n = 4) (1 : 100 (Tag-2A12)) (Bioprocessing Technology Institute, Singapore) (note: anti-ZO-1 (n = 4), 1 : 200 (ZO1-1A12, Alexa Fluor 488, Thermo Fisher Scientific, Rochester, NY, USA), was only incubated for 3 hours at RT, washed, and analyzed microscopically as it was already conjugated with Alexa Fluor). The samples (except ZO-1) were incubated with goat anti-mouse fluorescein isothiocyanate- (FITC-) conjugated secondary antibody in 5% BSA for 2 hours at RT. After each step, the cells were washed 3 times with 1X PBS. After removing the wall of the Lab-Tek slide, the cells were covered with mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) and covered with the cover slips. The Nikon Eclipse Ti-E (Nikon, Burgerweeshuispad, Amsterdam) microscope was used with NIS Elements software (Nikon) to observe the expression of the proteins and obtain images.