Rabbit anti cd86

Total protein was isolated from cultured cells using radioimmunoprecipitation assay lysis buffer (Thermo Fisher Scientific, USA) at 4 ​°C for 30 ​min. Lysates were centrifuged at 12,000×g for 20 ​min, and the supernatants were collected. Proteins were separated via 8% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to 0.22-μm polyvinylidene difluoride membranes (Millipore, USA). The membranes were blocked with 5% skim milk for 1 ​h and incubated at 4 ​°C overnight with the primary antibodies rabbit anti–NF–κB p65 (1:1000, Cell Signaling Technology, USA), rabbit anti-phospho–NF–κB p65 (1:1000, Cell Signaling Technology, USA), rabbit anti-inducible nitric oxide synthase (iNOS) (1:1000, Affinity Biosciences, China), rabbit anti-CD86 (1:1000, Abcam, USA) and rabbit anti-GAPDH (1:1000, Cell Signaling Technology, USA). After washing, the membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies for 1 ​h. Bands were visualized using an electrochemical luminescence reagent and the ChemiDoc XRS imaging system (Bio-Rad, USA).

To evaluate the anti-inflammatory,
myocardial structure, and angiogenesis after different treatment.
The heart sections from MI areas were fixed in 4 wt % paraformaldehyde
at 24 h for inflammation detection, and myocardial structure and angiogenesis
was detected at 4 weeks after hydrogel injection. Briefly, the fixed
hearts were dehydrated by gradient ethanol dehydration, made transparent
by xylene, embedded in paraffin, and sectioned to obtain the 4 μm
heart sections. For immunofluorescence staining, heart sections were
permeabilized in 0.1% Triton X-100 for 30 min, and then blocked with
bovine serum for 30 min. Primary antibodies were used to incubated
the heart sections overnight at 4 °C for detection of M1 macrophage
(rabbit anti-CD86 Abcam). Subsequently, the sections were washed with
PBS and then incubated with secondary antibodies (Cy3-labeled, goat
antirabbit IgG, BosterBio) for 90 min. Cell nuclei were stained with
DAPI. At the same time, for myocardial structure evaluation, the heart
sections were incubated with c-TnT and Cx43 antibodies. To evaluate
the new vessels forming, vWF and anti-α-smooth muscle actin
antibodies were used to incubate heart sections. Immunofluorescence
staining of all heart sections was then visualized using CLSM.

Cells were fixed in 4% formalin solution for 30 ​min and blocked with 5% bovine serum in 1 ​× ​PBS at 4 ​°C for 3 ​min. The primary antibodies rabbit anti-CD86 (1:200, Abcam, USA), rabbit anti-iNOS (1:200, Abcam, USA), rabbit anti–NF–κB p65 (1:200, Cell Signaling Technology, USA) and rabbit anti-phospho–NF–κB p65 (1:200, Cell Signaling Technology, USA) were applied to samples at 4 ​°C overnight and washed off thereafter. The samples were incubated with secondary antibodies (1:1000, Alexa Fluor 488 goat, Beyotime, USA) and FITC-phalloidin at 37 ​°C for 2 ​h. The cells were counterstained with DAPI and washed before imaging under an upright microscope.

Tissue sections were rehydrated and boiled in sodium citrate buffer for about 20 ​min for immunofluorescence staining. The primary antibodies rabbit anti-CD86 (1:200, Abcam, USA), rabbit anti-iNOS (1:200, Abcam, USA), anti-keratin 10 (K10; 1:200, Abcam, USA), rabbit anti-CD206 (1:800, Abcam, USA) and mouse anti-CD68 (1:1000, Abcam, USA) were incubated with samples at 4 ​°C overnight followed by washing. Sections were incubated with secondary antibodies for 2 ​h at room temperature. DAPI was used to stain nuclei. The sections for immunohistochemical staining were subjected to epitope retrieval in sodium citrate solution. After washing in PBS, the sections were incubated with rabbit anti-AGEs (1:200, Abcam, USA) as a primary antibody overnight at 4 ​°C. Images were evaluated using an upright microscope.