Rat anti mouse f4 80 monoclonal antibody

The muscle biopsy specimens from DMD patients and non-DMD controls were freshly frozen in liquid nitrogen–cooled isopentane. The frozen muscle Sects. (8 μm) were stained with HE and pathological changes were observed under a light microscope. IHC assay was according to the previous manufacturer's suggestion [25 (link)]. Briefly, the dry slides were preblocked in PBS containing 10% normal goat serum and incubated overnight with the primary antibodies for macrophages (rat monoclonal anti-mouse F4/80 antibody, Abcam, Cambridge, UK), CD4 positive T cells (mouse monoclonal antibody against CD4, Maxim, Fuzhou, China) or CD8 positive T cells (mouse monoclonal antibody against CD8, Maxim, Fuzhou, China), respectively. Then, the cells were rinsed and incubated with the appropriate secondary antibody (Proteintech, Wuhan, China) at 20 ℃ for 20 min. 3,3-diaminobenzedine tetrahydrocloride (Solarbio, Beijing, China) was used as chromogenic substrate. Lastly, the cells were counterstained with haematoxylin and mounted. The Ab binding was observed under a microscope.

Immunohistochemical analysis was performed on formalin-fixed, paraffin-embedded sections using following antibodies and dilution ratios: rat monoclonal anti-mouse F4/80 antibody (1:250, Abcam, Cambridge, UK), rabbit polyclonal anti-mouse CD3 antibody (1:500, Abcam), and rat monoclonal anti-mouse E-cadherin (1:200, eBioscience). Briefly, after dewaxing and rehydration, a heat-inducing antigen retrieval procedure using citrate buffer at pH 6.0 for 21 min was performed on all tissue sections, with subsequent washing in PBS and endogenous peroxidase blocking with EnVisionTM FLEX Peroxidase-Blocking Reagent (Dako, Agilent, Santa Clara, CA, USA) for 10 min. Sections were incubated with primary antibodies overnight. Sections stained with anti-F4/80 and anti-E-cadherin antibody were incubated with secondary polyclonal rabbit anti-rat immunoglobulins/HRP (1:500, Dako) for 60 min. The CD3 sections were treated by applying the commercial EnVisionTM FLEX/HRP detection reagent (Dako). Immunoreactions were developed with diaminobenzidine (DAB, Dako) diluted in EnVisionTM FLEX Substarte Buffer (Dako). The sections were counterstained with haematoxylin.

48 hours following IVC ligation, the formed thrombi were isolated and fixed in zinc formation. Cellular components of thrombi were determined using corresponding antibodies on paraffin-embedded sections. Macrophages were identified with a rat anti-mouse F4/80 monoclonal antibody (1:100) (Abcam, Cambridge, MA). Neutrophils were identified with a rabbit anti-mouse myeloperoxidase (MPO) polyclonal antibody (1:200) (DAKO, Carpinteria, CA). The NET-related markers cathelicidin-related antimicrobial peptide (CRAMP) and citrullinated histone H3 (Cit-H3) were detected using a rabbit anti-mouse CRAMP polyclonal antibody (1:200) (Innovagen, Lund, Sweden) and a rabbit anti-mouse Cit-H3 polyclonal antibody (1:100) (Abcam, Cambridge, MA) as described previously^{42 (link)}. Positive cells were detected with corresponding biotin-conjugated secondary antibodies. Stained cells were counted manually from five positively stained fields in each section using NIH ImageJ software and expressed as a percentage of positive cells per unit area.