Alexa fluor 568 conjugated donkey anti mouse secondary antibody

Tissue samples taken from scar tissue were formalin-fixed overnight at 4°C and transferred to 20% sucrose (overnight at 4°C) and then 30% sucrose (72 h at 4°C). The samples were then embedded in Tissue-Tek O.C.T. compound (Sakura Finetechnical Co., Japan) and sectioned on a cryostat set at 10-μm thickness (Leica Microsystems GmbH, Germany). The slides were fixed in acetone for 15 min at 4°C and then incubated in phosphate-buffered saline (PBS) containing 5% bovine serum albumin (BSA) with 0.1% Triton X-100 for 1 h at 25°C room temperature . The samples were incubated with primary antibodies overnight at 4°C: mouse monoclonal antibody against neurofilament 200 kDa (NF, 1:200, ab3966, Abcam, UK), S100 protein (1:100, ZM-0224, ZSGB-Bio, China), βIII tubulin (Tuj-1, 1:500, 05-559, Millipore, USA), nestin (1:500, MAB353, Millipore), CSPG (1:500, ab11570, Abcam), and vimentin (1:500, V6630, Sigma, USA). They were then incubated with Alexa Fluor 568-conjugated donkey anti-mouse secondary antibody (A10037, 1:800, Invitrogen, USA) for 1 h at RT. Cell nuclei were stained with Hoechst 33342 (1:1000, B2261, Sigma), and all images were taken with a Leica TCS SP8 confocal microscope.