Journal: Developmental biology

Article Title: The Transition from Proliferation to Differentiation Is Delayed in Satellite Cells from Mice Lacking MyoD

doi: 10.1006/dbio.1999.9284

Figure Lengend Snippet: Micrographs of myofiber cultures from control wildtype mice (A, B, and C) and MyoD−/− mice (D and E) reacted via double immunofluorescence with monoclonal antibodies against the nuclear antigen PCNA, MyoD, or myogenin along with the polyclonal antibody against ERK1/ERK2. All micrographs depict cultures that were maintained in basal medium + FGF2 at 2 ng/ml. (A and A′) A day 4 culture reacted with anti-PCNA and anti-ERK. (B and B′) A day 3 culture reacted with anti-MyoD and anti-ERK. (C and C′) A day 4 culture reacted with anti-myogenin and anti-ERK. (D and D′) A day 3 culture reacted with anti-PCNA and anti-ERK. (E and E′) A day 5 culture reacted with anti-myogenin and anti-ERK. (A″, B″, C″, D″, and E″) Parallel micrographs of DAPI stain which highlight all myofiber and satellite cell nuclei. For each antibody combination, reactivity with the monoclonal antibody was visualized with a fluorescein-labeled secondary antibody and reactivity with the anti-ERK antibody was visualized rhodamine-labeled secondary antibody. Arrows in parallel images point to the same locations of cells as identified by immunostaining and by DAPI. Note that not all positive nuclei or cells on the myofibers are in the same focal plane. Bar, 34 μm.

Article Snippet: A mouse monoclonal antibody against PCNA was from Boehringer Mannheim (Indianapolis, IN).

Techniques: Control, Immunofluorescence, Bioprocessing, Staining, Labeling, Immunostaining

Journal: Developmental biology

Article Title: The Transition from Proliferation to Differentiation Is Delayed in Satellite Cells from Mice Lacking MyoD

doi: 10.1006/dbio.1999.9284

Figure Lengend Snippet: Temporal appearance of cells or nuclei positive for ERK1/ERK2 and PCNA (A), MyoD (B), and myogenin (C) in cultures of myofibers isolated from Balb/C mice. Cultures were maintained in basal medium ± FGF2 at 2 ng/ml and the medium was changed daily. Plates were collected every 24 h and reacted via double immunofluorescence with the polyclonal antibody against ERK1/ERK2 in combination with the monoclonal antibodies against PCNA, MyoD, and myogenin. Immunostaining was performed as detailed in the legend to Fig. 1. Two parallel 35-mm plates were analyzed for each time point. For each panel, the total number of ERK+ cells and the number of doubly positive cells (PCNA+/ERK+, MyoD+/ERK+, myogenin+/ERK+) were determined. Nuclei positive for PCNA, MyoD, or myogenin which were not within ERK+ cells were never detected. The total number of ERK+ cells, shown in A, was similar for all three panels. Cells were scored as the number of positives on each individual fiber, analyzing 30 fibers per plate. Total positives were then averaged for duplicate plates and this value was eventually expressed per 10 fibers as indicated on the y axis. The error bar depicts the range of the variation between the duplicate plates of individual experiments.

Article Snippet: A mouse monoclonal antibody against PCNA was from Boehringer Mannheim (Indianapolis, IN).

Techniques: Isolation, Immunofluorescence, Bioprocessing, Immunostaining

Journal: Developmental biology

Article Title: The Transition from Proliferation to Differentiation Is Delayed in Satellite Cells from Mice Lacking MyoD

doi: 10.1006/dbio.1999.9284

Figure Lengend Snippet: Temporal appearance of cells or nuclei positive for ERK1/ERK2 and PCNA (A) and ERK1/ERK2 and myogenin (B) in cultures of myofibers isolated from MyoD−/− mice. Cultures were maintained in basal medium (±FGF2 at 2 ng/ml) and the medium was changed daily. Plates were collected every 24 h and reacted via double immunofluorescence with the polyclonal antibody against ERK1/ERK2 in combination with the monoclonal antibodies against PCNA and myogenin. Immunostaining was performed as detailed in the legend to Fig. 1. Two parallel 35-mm plates were analyzed for each time point. For each panel, the total number of ERK+ cells and the number of doubly positive cells (PCNA+/ERK+ or myogenin+/ERK+) were determined. Nuclei positive for PCNA or myogenin which were not within ERK+ cells were never detected. Also, immunostaining with the antibody against MyoD could not detect immunostained nuclei or cells. Cells were scored as the number of positives on each individual fiber, analyzing 30 fibers per plate. Total positives were then averaged for duplicate plates and this value was eventually expressed per 10 fibers as indicated on the y axis. The error bar depicts the range of the variation between the duplicate plates of individual experiments.

Article Snippet: A mouse monoclonal antibody against PCNA was from Boehringer Mannheim (Indianapolis, IN).

Techniques: Isolation, Immunofluorescence, Bioprocessing, Immunostaining