S 2000

The paraffin sections were baked at 65 °C overnight. Slides were then deparaffinized and rehydrated. Dako endogenous blocking reagent (S2003, Dako, Carpinteria, CA, USA) was then used to quench endogenous peroxidase for 15 min. Non-specific binding sites were blocked with 1:10 normal horse/goat serum (S-2000, Vector Laboratories, Burlingame, CA, USA) for 30 min at room temperature. Primary antibodies: 1:400 dilution of MMP13 (ab39012, Abcam, Cambridge, UK), 1:1 000 dilution of ColX (ab49945, Abcam, Cambridge, UK); 1:400 dilution of Admts4 (ABT178, Millipore, Billerica, MA), and 1:500 dilution of Adamts5 (ab41037 Abcam, Cambridge, UK) were added, and the slides were incubated at 4 °C overnight. For IHC assays, the secondary biotinylated goat anti-mouse antibody (BA-9200, Vector Laboratories) at the dilution of 1:200 was added for 30 min on the second day, followed by incubation with 1:250 streptavidin (21130, Pierce, Rockford, IL, USA) for 30 min. Positive staining was detected by Romulin AEC Chromagen (Biocare Medical RAEC810L, Concord, CA, USA). For IF staining, an appropriate secondary antibody conjugated to a fluorescence probe was added, incubated at room temperature for 1 h, rinsed in PBS, and mounted in an anti-fading mounting media (Vector Laboratories, Burlingame, CA). Results were obtained using an Olympus BX43 upright microscope (Olympus Optical, Tokyo, Japan).

Immunohistochemistry analysis was performed on 10 µm paraffin embedded serial sections from the enucleated mouse eyes as described in our previous studies [72 ]. At minimum 100 µm of retina/sample was evaluated by IHC. Briefly, sections were blocked with 2% normal horse serum (#S-2000 VectorLabs, CA) in PBS, and incubated with the following cell type-specific primary antibodies in a 1:200 dilution: rhodopsin (mouse monoclonal, Millipore MAB5356); green/red opsin (rabbit polyclonal, Millipore AB5405); blue opsin (rabbit polyclonal, Millipore AB5407); GFP (1:500, rabbit polyclonal, Abcam ab290). The following day, sections were rinsed with PBS and incubated with the corresponding secondary antibody (1:400 Alexa fluor 488 goat antirabbit, Invitrogen A11008) and nuclei were stained with 4,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI). Over 500 μm of sections/animal were visualized and representative images of IHC labeling were captured using a Leica DMI6000 fluorescent microscope equipped with the appropriate bandpass filter for each fluorochrome. Cell counts were performed in a double-blinded manner over 100 μm retinal area (N = 10/strain/experimental group).

Heart samples were fixed in 4% paraformaldehyde (sc‐281,692, Santa Cruz) and were embedded in paraffin. Sections were cut (8 μm) and re‐hydrated through sequential incubations in a series of graded xylene (X3P‐1GAL, Fisher Scientific), ethanol (BP2818, Fisher Scientific), and phosphate buffer solution (PBS). Antigen retrieval was performed by running slides submerged in IHC‐TekTM Epitope Retrieval Solution. Slides were blocked with 15% horse serum (S‐2000, Vector Laboratories) for 1 h and followed by incubation with anti‐MPO antibody (1:400, ab208670, Abcam). DAB labeling was performed with HRP‐conjugated donkey anti‐rabbit IgG (1:250, A10040, Invitrogen). Hematoxylin (H‐3404, Vector Laboratories) was used for nuclear staining. For the result quantification, 10 pictures were taken from each sample and the average number of cells per picture was calculated.

All slides were manually processed. Sections were deparaffinized in xylene, placed in citric acid-based retrieval buffer (pH 6.0), and heated at 110 °C for 15 min in a pressure cooker, according to the manufacturer’s protocol. The slides were washed in distilled water, treated with 3% H₂O₂ for 20 min, washed three times in 0.1% Triton X-100/PBS, and then blocked using 1.5% serum solution/PBS. Antibodies were diluted in PBS. Samples were incubated with primary antibodies overnight at 4 °C, washed three times, incubated with secondary antibodies for 20 min at room temperature, and then washed three times. Detection was performed using an avidin-biotinylated enzyme complex kit (Vectastain ABC Kit, PK6100, Vector, Newark, USA) and 3ʹ-3-diaminobenzidine (DAB) substrate (Wako, Japan), following the manufacturer’s protocol. Slides were counterstained with hematoxylin, washed, dehydrated with ethanol, and mounted. The primary antibodies were CD21 (2G9, NCL-L-CD21-2G9; Leica Biosystems, Germany), CD23 (SP23, ab16702; Abcam, Cambridge, UK), PD-1 (NAT105, ab52587; Abcam, Cambridge, UK) and the secondary antibodies were horse anti-mouse IgG (BA-2000; Vector, Newark, USA) and goat anti-rabbit IgG (BA-1000; Vector, Newark, USA). The serum-blocking solutions were horse (S-2000; Vector, Newark, USA) and goat (S-1000; Vector, Newark, USA).

The HAECs were plated on glass-bottom dishes precoated with gelatin. After pretreating with their designated DOX and/or VitD3 dose, the HAECs were rinsed with PBS, fixed with 4% formaldehyde, and permeabilized with 0.3% Triton X-100 using the BD cytofix/cytoperm fixation/permeabilization kit (BD biosciences, 554714). After rinsing with PBS twice and being blocked with 10% normal horse serum (Vector Laboratory, S-2000) in PBS for 1 hour, the HAECs were incubated with primary antibodies (diluted with 10% horse serum in PBS) overnight at 4°C. This was followed by a wash and exposure to a 10% horse serum in PBS diluted fluorescently labeled secondary antibodies (Vector Laboratory, DI-1088, DI-1094 or/and DI-2549) for one hour in the dark at room temperature. After completely washing with PBS and mounting with VECTASHIELD® Antifade Mounting Medium with DAPI (Vector Laboratory, H-1200), the fluorescence of HAECs was examined with a Leica laser point scanning confocal microscope (Leica TCS SPE).