Alexa fluor 555 goat anti mouse igg

After the rats were initially perfused with PBS solution, a subsequent perfusion was conducted using a 4 % formaldehyde solution. A 2 cm long section of spinal cord tissue from the injury epicenter was continued to be fixed in 4 % formaldehyde at 4 °C for 24 h. The tissue was dehydrated in a sequential manner in 10 %, 20 %, and 30 % sucrose solutions, each for one day. Subsequently, the tissue was embedded in optimal cutting temperature (OCT) compound and stored at −20 °C. Afterward, using a frozen microtome (CM30505 Leica), the spinal cord was cut into 6 μm thick cryosections. All cryosections were washed for 5 min in 1 × PBS to remove OCT. Then, the cryosections were blocked with PBS containing 0.25 % Triton X-100 (Cat# T8200; Solarbio) and 5 % BSA (Cat# A8020; Solarbio) at RT for 1 h.
GFAP (1: 500; Cat# ab279289; Abcam) and NeuN (1: 500; Cat# ab177487; Abcam) were selected as primary antibodies with an incubation time of 2 h at RT. After circling the appropriate range on the slide with a PAP pen, the primary antibodies were added and incubated for 2 h at RT. Goat Anti-Mouse IgG (Alexa Fluor® 555) (1: 400; Cat# ab150118; Abcam) and Goat Anti-Rabbit IgG (Alexa Fluor® 488) (1: 400; Cat# ab150081; Abcam) were used as secondary antibodies for incubation. After washing, sections were covered and slipped with a mounting medium containing DAPI (Cat# ab104139; Abcam).

Cells were seeded on a coverslip and cultured for 3 to 5 days before being fixed in 4% paraformaldehyde for 20 minutes. The cells were permeabilized with 0.1% Triton X-100 for 5 minutes when needed. Cells were then blocked in 5% BSA in PBS for 30 minutes; all these procedures were performed at room temperature. Cells were then incubated overnight at 4 °C with primary antibodies. For pluripotent stem cells, anti-SSEA4 (1:200 dilution; Abcam, UK) and anti-Nanog (1:200 dilution; Abcam) were applied. For cardiomyocytes, anti-α-actinin (1:200 dilution; Sigma-Aldrich), anti-cardiac troponin I (1:200 dilution; Abcam), and anti-Nav1.5 (1:100 dilution; Abcam) were used. The following secondary antibodies included Alexa Fluor 488 goat anti-rabbit IgG (1:800 dilution; Abcam) and Alexa Fluor 555 goat anti-mouse IgG (1:800 dilution; Abcam) were incubated with samples at room temperature for 30 minutes. The nuclei were stained with DAPI (Life Technologies, USA). Then, the cells were rinsed with PBS and observed under a fluorescence microscope (Axio Imager A2, Carl Zeiss, Germany). Images were analyzed and merged with ImageJ (NIH, version 1.8.0_77).