Rat anti mouse cd68

After invivo fluorescence imaging, C57BL/6 mice were euthanized and the limb muscles containing femoral arteries and paw tissues were dissected. The tissues were immersed in tissue freezing medium (TFM, Triangle Biomedical Sciences), frozen, and sectioned in 8 µm segments. The tissue sections were rinsed with PBS and fixed with 4 % paraformaldehyde for 20 min at room temperature (RT). After an additional series of washes with PBS, the tissue sections were cleared with 3 % sodium deoxycholate solution for 2 hours at RT, blocked with 20 % normal goat serum in 1 % BSA-PBS for 2 hours at 37°C, incubated with rat anti-mouse CD169 (1:200, Serotec) or rat anti-mouse CD68 (Abcam) at 4°C overnight (> 17 hours) diluted in 1 % BSA-PBS, rinsed three times with PBS, and incubated with Alexa Fluor 594 goat anti-rat IgG secondary antibody at RT for 2 hours. Then, the slides were washed with PBS several times, counterstained with Hoechst 33342 and mounted with ProLong Gold antifade reagent (Life Technologies). The images were captured with an Eclipse Ti-E inverted microscope (Nikon) modified for NIR fluorescence detection.

Mice were intracardially perfused with heparinized saline and 4% phosphate-buffered PFA, and the brain and liver were harvested. The organs were cryoprotected in 20% PBS sucrose for 24 hours and embedded in a Cryomatrix (Tissue-Tek OCT) before cryostat sectioning. Cryostat-cut sections (thickness, 10 μm) were collected on polylysine slides and stored at –80°C before processing. Sections were coincubated overnight with rat anti-mouse CD68 (1:800; Abcam, 53444), goat anti-mouse collagen IV (1:1000; SouthernBiotech, 1340), and rat anti-mouse VCAM-1 (1:500; BD Bioscience, 550547). Primary antibodies were revealed by using Fab′2 fragments of donkey anti-rabbit linked to Cy3, anti-rat linked to Cy3, and anti-goat IgG linked to FITC (1:600; Jackson ImmunoResearch, West Grove, USA). Washed sections were coverslipped with antifade medium containing DAPI. Epifluorescence images (~10 images per section) were digitally captured using a Leica DM6000 epifluorescence microscope coupled CoolSNAP camera, visualized with Leica MM AF 2.2.0 software (Molecular Devices, USA), and further processed using ImageJ 1.51k software.

For immunohistochemical analysis of aortic root sections, tissues that were frozen in OCT compound (Tissue-Tek, Torrance, CA) were serially cut in 8 μm thick sections, covering a length of approximately 640 μm from the aortic sinus (where the aortic valve leaflets appear) to the distal region of the root. Sections were mounted on glass slides and fixed in 4% paraformaldehyde for 30 min and treated with 0.1% Triton X-100 in PBS for 15 min. After blocking in 1% BSA/PBS at room temperature for 2 h, slides were incubated overnight at 4°C with a combination of rabbit anti-mouse SAA (catalog no.: ab199030; Abcam, Cambridge, MA) and rat anti-mouse CD68 (catalog no.: ab53444; Abcam, Cambridge, MA) diluted 1:200 in 1% BSA/PBS for each primary antibody. After washes with PBS, SAA was detected using Alexa Fluor 488-labeled goat anti-rabbit IgG (1:200 dilution; Molecular Probes; Cambridge, MA), and CD68 was detected using Alexa Fluor 568-labeled goat anti-rat IgG (1:200 dilution; Molecular Probes). Slides were mounted using fluorescence-protecting medium containing 4′,6-diamidino-2-phenylindole (Vectashield; Vector Laboratories). Images were captured by fluorescence microscopy (Nikon Eclipse 80i microscope, Nikon Instruments), and areas of immunopositive staining were quantified using Nikon NIS-elements software.

Mouse anti-rat CD11b/c PerCP-eFluor^® 710 (Cat#46–0110) and its isotype control (Cat#46–4724) for flow cytometry were purchased from eBioscience (San Diego, CA). Anti-Mouse Ig APC (Cat# 550826) for flow cytometry was purchased from BD bioscience (San Jose, CA) and Donkey Anti-Rabbit IgG PE (Cat#12-4739-81) was from eBioscience (San Diego, CA). Anti-rat CD68 FITC (Cat#SM1550F) for flow cytometry was from Acris-antibodies (San Diego, CA). Rabbit anti-rat CD206 (Mannose Receptor antibody) (Cat#ab64693), mouse anti-rat CD11c (Cat#ab11029) and mouse anti-rat CD68 (Cat#ab53444) for immunohistochemical staining were from Abcam (Cambridge, MA).

After the last MRI scan, each animal were sacrificed, knee joints were resected and placed in Cal-Ex II (Fisher Scientific) for 4–5 days. Cal-Ex II is a mixture of formaldehyde and formic acid, which decalcifies the bones and fixes the tissue simultaneously. Decalcified specimen were dissected parasagittally, dehydrated through graded alcohol washes, embedded in paraffin, cut in 5 μm sections and stained with standard haematoxylin and eosin (H&E) to define the morphology of the implant and Alcian blue to confirm hyaline cartilage matrix production in the viable implants. DAB-Prussian blue stains and anti-CD68 stains were performed on deparaffinized sections similar to in vitro studies (see above). Anti-CD68 staining specific for ED-1 macrophages (Primary Antibody: Mouse anti-rat CD68, Abcam, Cambridge, MA, USA; Secondary Antibody: Alexa flour 647 goat anti mouse, Invitrogen, Eugene, OR, USA) was added to detect macrophages in the transplant and anti-Dextran, Clone DX1, FITC (Mouse Anti-Dextran, FITC-conjugated, Stem Cell Technology, Tokwila, WA, USA) to detect ferumoxytol labeled hMSC.