Alexa fluor 488 conjugated anti goat igg

Fresh LNs were embedded in tissue-freezing medium. Cryostat sections (8 μm thick) were cut for imaging by fluorescence confocal microscopy. The following primary Abs were used for tissue staining: anti-HEV MECA79 (sc-19602, SCBT), anti-Lyve-1 (ab14917, Abcam), anti-PDPN (AF3244, R&D Systems), anti-ER-TR7 (sc-73355, SCBT), anti-CD11b (101202, BioLegend), anti-PDGFRβ (136005, Biolegend), anti-NG2 (ab129051, Abcam), anti-RANKL (510002 Biolegend), anti-CD35 (NBP2-52667, Novus Biologicals), anti-MAdCAM (16-5997-85,eBioscience), anti-ZO-1 (61-7300, Invitrogen), anti-Collagen I (ab34710, Abcam), anti-Fibronectin (ab45688, Abcam), anti-LepR (L9536, Sigma-Aldrich). The following secondary Abs were used: Alexa Fluor 488-conjugated anti-rabbit IgG, Alexa Fluor 594-conjugated anti-rabbit IgG, Alexa Fluor 488-conjugated anti-rat IgG, Alexa Fluor 594-conjugated anti-rat IgG, Alexa Fluor 488-conjugated anti-goat IgG, and Alexa Fluor 594-conjugated anti-goat IgG (Jackson ImmunoResearch). The stained tissue sections were imaged using EVOS FL Auto 2 Imaging System (Thermo Fisher Scientific). For the quantification of images, all images were automatically processed using ImageJ (NIH) and split into RGB channels. Auto threshold was used to convert intensity values of the immunofluorescent stain into numeric data. DAPI (VECTASHIELD, Vector Laboratories) was used to stain the cell nuclei.

Immunofluorescent staining was performed according to the standard protocol as previously reported [19 (link)]. Briefly, the heart sections were incubated with anti-vimentin, anti-CD68, anti-CD31 (Abcam), and anti-α-SMA (Santa Cruz) to detect fibroblasts, macrophages, and blood vessels. Then, FITC-conjugated anti-mouse IgG or anti-rabbit IgG was added and incubated for 1 h before observation. For detection of neovascularization, 1 mg/mL Griffonia (Bandeiraea) simplicifolia lectin 1 (BS1 lectin; Vector) was injected into the left ventricle via direct cardiac puncture 15 min before the sacrifice of mice. Slides were sequentially stained with goat anti-BS1 lectin antibody (Vector) and Alexa Fluor 488-conjugated anti-goat IgG (Jackson ImmunoResearch). Finally, the heart sections were mounted with DAPI-containing anti-fade medium and imaged.