After two weeks, the rats were euthanized with an overdose of sodium pentobarbital (0.5 mL/kg). The calvarial defect region was harvested, fixed in 4% paraformaldehyde and embedded in paraffin wax. The samples were cut into 5 μm thick serial sections for subsequent histological evaluation. Haematoxylin and eosin (HE) staining and CD31 immunohistochemical assays were performed. Endomucin (Emcn)/CD31 immunofluorescence (IF) staining was performed as follows. Tissues sections were prepared from the groups. A rabbit anti-CD31 antibody (1:100, Bioswamp, China) and rat anti-Emcn antibody (1:100, Santa Cruz, USA) were used as the primary antibodies, and FITC-conjugated goat anti-rat IgG (1:200, Proteintech, USA) and goat anti-rabbit IgG-Alexa Fluor® 594 antibodies (1:200, Huabio, China) were used as the secondary antibodies. The stained tissue sections were mounted and imaged by fluorescence microscopy. Angiogenesis was quantified based on the percentage area of Emcn/CD31-positive microvessels in randomly selected fields of view.
Fitc conjugated goat anti rat igg
Manufactured by Proteintech
Sourced in United States
FITC-conjugated goat anti-rat IgG is a secondary antibody that binds to rat immunoglobulin G (IgG) and is conjugated with the fluorescent dye fluorescein isothiocyanate (FITC). It is used for the detection and localization of rat IgG in various immunological techniques, such as immunofluorescence, flow cytometry, and Western blotting.
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2 protocols using fitc conjugated goat anti rat igg
1
Histological Evaluation of Angiogenesis
2
Immunohistochemical Analysis of p-AMPKα and Mac-2
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Sections were immersed in HistoVT One (Nacalai) for antigen activation. Sections were permeabilized with 0.1% Triton X-100 in tris buffered saline for staining of intracellular proteins. Sections were blocked with 0.3% H 2 O 2 in methanol and Blocking One Histo (Nacalai). Sections were incubated with anti-p-AMPKα (T172) rabbit antibody (Cell Signaling Technology) and anti-Mac-2 rat antibody (Biolegend) at 4°C overnight. The secondary antibody was HRP-conjugated goat anti-rabbit IgG (Cappel) and HRP-conjugated rabbit antirat IgG (Abcam). The Peroxidase Stain diaminobenzidine kit (Nacalai) was used as a chromogenic agent.
For immunofluorescence staining of Gr-1-positive cells, sections were performed antigen activation, subsequently blocked with Blocking One Histo (Nacalai). Sections were incubated with anti-Gr-1 rat IgG (Biolegend) at 4°C overnight. The secondary antibody was FITC-conjugated goat anti-rat IgG (Proteintech). Sections were incubated with Vector TrueVIEW Autofluorescence Quenching Kit (Vector) for reducing the autofluorescence emission.
For immunofluorescence staining of Gr-1-positive cells, sections were performed antigen activation, subsequently blocked with Blocking One Histo (Nacalai). Sections were incubated with anti-Gr-1 rat IgG (Biolegend) at 4°C overnight. The secondary antibody was FITC-conjugated goat anti-rat IgG (Proteintech). Sections were incubated with Vector TrueVIEW Autofluorescence Quenching Kit (Vector) for reducing the autofluorescence emission.
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