Alexa fluor 488 conjugated anti rabbit igg h l antibody

Cells were stripped from plates and treated with 75 mM KCl solution at 37°C for 6 min. After centrifugation, the cells were fixed with ice-cold methanol/acetic acid (3:1). The fixed cells were dropped to a slide on the floor with a ~20° angle from the distance more than 140 cm. After air-drying in a fume hood, the slides were pretreated with 3.7% formaldehyde and unmasking solution (ProHisto, LLC). Cells were then dehydrated sequentially with 70, 85, and 95% ethanol solution. Alexa Fluor-546-conjugated (CCCTAA)₃ or (TTAGGG)₃ probes (IDT) was then added to slides and heated in 87°C oven for 10 min and cooled down to room temperature for 1 h. After being blocked, cells were immunostained with mMyh antibody, followed by Alexa Fluor-488-conjugated anti-rabbit IgG (H+L) antibody (Molecular Probes). The immunostained cells were then counterstained with DAPI and mounted with coverslips. Images were captured with a Nikon PCM 2000 confocal microscope scanning system.

Anti-goat IgG antibody conjugated to fluorescein isothiocyanate was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Rabbit polyclonal antibodies against ionized calcium-binding adapter molecule 1 (Iba1; Wako Pure Chemical Industries, Ltd., Osaka, Japan) and β-catenin (Sigma-Aldrich Co., St. Louis, MO, USA), goat polyclonal antibody against doublecortin (DCX; Santa Cruz Biotecchnology, Santa Cruz, CA), rat monoclonal antibody against 5-bromo-2′-deoxyuridine (BrdU; Abcam, Ltd., Cambridge, MA, UK), and mouse monoclonal antibodies specific for glial fibrillary acidic protein (GFAP; Sigma-Aldrich Co., St. Louis, MO, USA), nestin (Millipore Co., Boston, MA, USA), and neuronal nuclear antigen (NeuN; Chemicon International, Temecula, CA, USA) were used as primary antibodies. Alexa-Fluor 594-conjugated anti-rat IgG (H+L) antibody, Alexa-Fluor 488-conjugated anti-rabbit IgG (H+L) antibody, and Alexa-Fluor 488-conjugated anti-mouse IgG (H+L) antibody were obtained from Molecular Probes (Eugene, OR, USA). Lithium carbonate and TMT were supplied by Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All other chemicals used were of the highest purity commercially available.