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. 2012 Apr;31(3):187–196. doi: 10.1016/j.matbio.2011.12.003

Fig. 3.

Fig. 3

Immunofluorescence analysis of collagen VI α3 and α 6 chains in normal muscle cultures.

a: The collagen VI α3 chain (red fluorescence) is detected in the extracellular matrix of both proliferating and long-term (after ascorbic acid treatment for 7 days post-confluence) samples, however the arrangement of the collagen VI network appears better organized in long term cultures. Bar, 100 μm. Nuclei were counterstained with DAPI (blue).

b: Collagen VI α6 chain (red fluorescence) deposits are detectable in the extracellular matrix of muscle cultures after ascorbic acid treatment for 7 days post-confluence. α6-containing structures (arrows, left upper panel) show a filamentous arrangement, as detected at higher magnification (right upper panel). Lower panels show double labeling with antibodies against the anti-α6 (red fluorescence) and α3 (green fluorescence) chains. Filamentous α6 chain-containing structures (red) co-localize with α3 chain-containing positive microfilaments, as revealed by the merged image (lower, right panel), however, an independent α3 chain-containing network (green fluorescence) can be also detected. Bar, 20 μm. Nuclei were counterstained with DAPI (blue).

c: Real Time-PCR analysis of COL6A3 and COL6A6 transcripts. Reported values are the mean of two different experiments utilizing ACTB and GAPDH as reference transcripts. The relative ratios ofCOL6A3/Actin-GAPDH and COL6A6/Actin-GAPDH mRNAs in proliferating cells were normalized to 1. Both COL6A3 and COL6A6 transcript levels were increased in long-term cells (1.7:1 for COL6A6 and 2.9:1 for COL6A3).

d: Immunofluorescence analysis of collagen VI α3 and α6 chains in long-term normal muscle cell cultures in the absence of ascorbic acid treatment. Samples were double-labeled for desmin (upper panels, green fluorescence) and for α-smooth muscle actin (α-SMA; lower panels, green fluorescence). The collagen VI α3 chain (red fluorescence, left, upper panel) appears to be absent in desmin-positive cells and accumulates in desmin-negative cells, indicating that the fibroblasts are the main source of α3 chain. Similarly, the α6 chain (red, right, upper panel) is absent in desmin-positive, but different from the α3 chain, it is detected intracellularly only in a subset of desmin-negative cells. Cell producing collagen VI α6 chain (red) are positive for α-smooth muscle actin (green, right, lower panel), indicating that they could be myofibroblasts. Bar, 20 μm. Nuclei were counterstained with DAPI (blue).