Alexa fluor 488 conjugated donkey anti rabbit igg h l antibody

U87MG and U251MG cells stably expressing YKL-40 FL or SV were plated into 8-well chamber slides at a density of 8,000 cells per well. Forty-eight hours after seeding, the cells were washed twice with PBS, fixed with 4% formaldehyde solution in PBS for 15 min, and permeabilized with 0.5% Triton X-100 in PBS for 12 min. Permeabilized cells were blocked with blocking buffer (3% bovine serum albumin in PBS) for 1 hour at RT. The slides were incubated with anti-CHI3L1/YKL-40 (Abcam, #ab77528) and anti–protein disulfide isomerase (PDI) (Abcam, #ab2792) antibodies, followed by incubation with an Alexa Fluor 488-conjugated donkey anti-rabbit IgG (H + L) antibody (Thermo Fisher, #A-21206) and an Alexa Fluor 555-conjugated donkey anti-mouse IgG (H + L) antibody (Thermo Fisher, #A-31570). The nucleus was stained with 300 nM DAPI in PBS for 15 min at RT. Slides were mounted with mounting medium (VECTOR laboratories, #H-1200) and covered with large coverslips (24 mm × 50 mm). Cells were examined by confocal fluorescence microscopy (Olympus FV1200 SIM Confocal System with ZDC, Olympus). The fluorescence signals were quantified by Fiji-ImageJ software.

At the indicated period after the aneurysm induction, 5-µm-thick frozen sections were prepared. After blocking with 3% donkey serum (#AB_2337258, Jackson ImmunoResearch, Baltimore, MD, USA), slices were incubated with primary antibodies, followed by incubation with secondary antibodies conjugated with a fluorescence dye (Jackson ImmunoResearch). Finally, fluorescent images were acquired using a confocal fluorescence microscope system (FV1000 or FV3000, Olympus, Tokyo, Japan).
The following primary antibodies were used: mouse monoclonal anti-CD68 antibody (#ab31630, Abcam, Cambridge, UK), rabbit polyclonal anti-myeloperoxidase (MPO) antibody (#ab9535, Abcam), rabbit polyclonal anti-tumor necrosis factor (TNF)-alpha antibody (#ab6671, Abcam), mouse monoclonal anti-smooth muscle α-actin (SMA) antibody (#M0851, Dako, Agilent, Santa Clara, CA, USA).
The following secondary antibodies were used; Alexa Fluor 488-conjugated donkey anti-mouse IgG H&L antibody (#A21202, Thermo Fisher Scientific, Waltham, MA, USA), Alexa Fluor 488-conjugated donkey anti-rabbit IgG H&L antibody (#A21206, Thermo Fisher Scientific), Alexa Fluor 594-conjugated donkey anti-mouse IgG H&L antibody (#A21203, Thermo Fisher Scientific).