Alexa 488 green

After terminal anesthesia by barbiturate overdose, mice were perfused transcardially with a phosphate buffered saline rinse followed by either 4% paraformaldehyde or 10% formalin. Spinal cords were removed, post-fixed overnight, and cryoprotected in buffered 30% sucrose for 48 hours. Frozen sections (30 μm) were prepared using a cryostat microtome (Leica) and processed for immunofluorescence as described7 (link)12 (link)13 (link). Primary antibodies were: rabbit anti-GFAP (1:1000; Dako, Carpinteria, CA); rat anti-GFAP (1:1000, Zymed Laboratories); sheep anti-BrdU (1:300, Maine Biotechnology Services, Portland, ME); rat anti-CD133 (1:200; Millipore, Temecula, CA); and rat anti-vimentin (clone # 280618; 1:150; Novus Biologicals, Littleton, CO). Fluorescence secondary antibodies were conjugated to: Alexa 488 (green) or Alexa 405 (blue) or to Cy3 (550, red) or Cy5 (649, far red) all from Jackson Immunoresearch Laboratories. Nuclear stain: 4′,6′-diamidino-2-phenylindole dihydrochloride (DAPI; 2 ng/ml; ThermoFisher). Sections were coverslipped using ProLong Gold anti-fade reagent (InVitrogen, Grand Island, NY). Sections were examined and photographed using deconvolution fluorescence microscopy and scanning confocal laser microscopy (Zeiss, Oberkochen, Germany).

Immunofluorescence was performed according to our previously published work[49 (link)].The following antibodies were used for immunoinfluscent assay: anti-K24 (1:200, Abcam, ab87195), α-tubulin (1:200, Santa Cruz, sc-5286). Secondary antibodies labeled with Alexa 488 (green) and cy3 (red) (1:5000, Jackson ImmunoResearch) were used. Cell nuclei were stained with DAPI (Roche) for 10 minutes at a concentration of 5ug/ml (applied after incubation with secondary antibody). Negative controls stained with secondary antibody alone or Rabbit IgG showed no immunolabelling. The K24 location in keratinocytes and epidermis were detected using an inverted fluorescence microscope (Leica, German). The α-tubulin cytoskeletons of lentivirus-infected keratinocytes were visualized using a confocal laser scanning microscopy (Leica, SP8, Germany).