Alexa fluor 488 goat anti rat

Femur samples were fixed in 4% paraformaldehyde (Sigma) for 4 h and equilibrated in 30% sucrose (Sigma)/PBS. After two rinses with cold PBS, samples were decalcified with 0.25 M EDTA (Acros) for 2 days and then embedded in O.C.T. (Sakura). For staining, monoclonal antibodies against VCAM-1 (BD Pharmingen) and CXCL-12 (BD Pharmingen), polyclonal antibodies against IL-7 (Abcam) and IL-15 (Abcam), and cell labeling reagents (Molecular Probes), including 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester (5(6)-CFDA, SE; CFSE), CellTracker™ Red CMTPX Dye, and DAPI (4′,6-diamidino-2-phenylindole), were used. For secondary antibodies, Alexa Fluor 488 goat anti-rabbit and Alexa Fluor 488 goat anti-rat were purchased from Abcam. Cryostat sections were carried out in Leica CM1900.

For slice immunofluorescence, the slices were firstly deparaffinized and rehydrated, and antigen retrieval was done. For cell immunofluorescence, cells were fixed with 4% paraformaldehyde. For both slice and cell immunofluorescence, samples were permeabilized, blocked, and incubated in primary antibody rabbit anti-mouse HIF-1α (1:200, Immunoway, Plano, TX), rat anti-mouse CD44 (1:250, Abcam, Cambridge, United Kingdom), rabbit anti-mouse POSTN (1:600, Abcam, Cambridge, United Kingdom) at 4°C overnight. Then, the samples were washed and incubated in Alexa Fluor 555 goat anti-rabbit (1:500, Invitrogen, Waltham, MA) and Alexa Fluor 488 goat anti-rat (1:1000, Abcam, Cambridge, United Kingdom) secondary antibody at room temperature for 1 h. They were stained with DAPI (Beyotime Biotechnology, Shanghai, China) for 10 min. The samples were photographed using confocal laser-scanning microscopy (Leica, Germany), and semi-quantification of intensity was analyzed through Image J.

For flow cytometry, BV480-anti-CD105 (MJ7-18) and BV650-anti-CD31 (390) from BD Bioscience; APC-CD140a (APA5), PE-Cy5-anti-Ter119 (Ter119), PE-Cy5-anti-CD45 (30-F11), and AF700-anti-Ly6A/E (D7), all from eBioscience; FITC-anti-Ter119 (Ter119), FITC-anti-CD45 (30-F11), FITC-anti-CD31 (390), from Biolegend; and FITC-anti-Endomucin (EMCN) from Santa Cruz were used.
For immunofluorescence staining, the following primary antibodies were used: rabbit anti-mouse CD31, rabbit anti-mouse Aggrecan, rabbit anti-mouse Osterix, and rabbit anti-mouse periostin all from Abcam; and rat anti-mouse EMCN from Santa Cruz; the secondary antibodies were as follows: goat-anti-rabbit-Alexa Fluor 555, goat-anti-rat-Alexa Fluor 488, and goat-anti-rabbit-Alexa Fluor 488, all from Abcam. Prolong gold antifade reagent with DAPI from CST was used.

To perform immunofluorescence staining, brain sections were treated with dewaxing and tissue antigen recovery, then permeabilized with 0.2% Triton X‐100 and blocked in 10% normal goat serum for 1 h at 37°C. After blocking, brain sections were incubated with primary antibodies recognizing CD3, CD8a, NeuN, IBA‐1, CCR2, Ki‐67, CCL‐2, PD‐1, PD‐L1, and STAT‐1 in 5% nonfat dry milk for overnight at 4°C. Sections were washed with PBS with 0.2% Tween‐20 (PBST) 3 times for 5 min at 20°C. Corresponding secondary antibodies where goat anti‐rat‐PE (Southern Biotech, Cat.3050‐09), Goat anti‐rabbit‐PE (Abcam, Cat.ab72465), goat anti‐rabbit‐dylight 488 (Rockland, Cat.611‐141‐002), and goat anti‐rat Alexa Fluor 488 (Abcam, Cat.ab150165) in PBST for 1 h at 37°C. After washing with PBST, the nuclei were stained with DAPI (Abcam, Cat.ab104139) for 15 min at 20°C, and a mounting medium (Life Technologies) was employed to cover the sections. Slides were examined by a fluorescence microscope (Olympus BX51), and images were captured with Caseviewer software. The mean fluorescence intensity was calculated by ImageJ software (version 1.53).

The incubation and washing steps were the same as described above. The MLH1 immunodetection was with mouse anti-MLH1 (1:100), followed by the secondary goat anti-mouse antibody conjugated with Alexa Fluor 555, (red fluorescence) (Abcam plc, Cambridge, CB2 0AX, UK). The MUS81 immunodetection was with guinea pig anti-MUS81 (1:100), followed by the secondary goat anti-guinea pig Alexa Fluor 555 (red fluorescence) (Abcam plc, Cambridge, CB2 0AX, UK). Simultaneous ZYP1 immunodetection was with rat anti-ZYP1 (1:20), followed by goat anti-rat Alexa Fluor 488 (green fluorescence) (Abcam plc, Cambridge, CB2 0AX, UK).