1984b). 5% (w/v) bovine serum albumin in TBST (20 mm Tris, 140 mm NaCl and 0.1% Tween20 pH 7.6) for 1 h prior to probing with the primary antibody (diluted in blocking buffer) for 12 h. Subsequently, the membrane was washed three times with TBST and was then incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. This was followed by TBST wash to remove any unbound secondary antibody. Immunoreactivity was visualised using enhanced chemiluminescence. Multiple exposures (3C30 s) were taken to obtain sub-saturation band intensities. The X-ray films were scanned using a Bio-Rad Gs-710 imaging densitometer. Band intensities from scanned images Ac-Gly-BoroPro of Western blots were quantified using Quanti-Scan software from BioSoft (version Windows 3.x, Cambridge, UK). Band intensity was normalised to alpha tubulin abundance. Statistical analysis Two-way anova was performed to examine the effect of genotype (wild type vs. myostatin null) and the effect of age (4 months old vs. 19 months old) on each parameter. showed densely packed aligned fibres (Fig. 1A,E). In contrast, in animals showed a thick honeycomb organisation. In contrast, the connective tissue fibres from TA shows a mixture of dense and fine fibres. (B) Four-month-old TA shows a dense honeycomb pattern of connective tissue. (D) Nineteen-month-old BB shows a densely arranged regular network of collagen fibrils dense fibre. (F) Four-month-old 0.001). We measured the thickness of the endomysium and perimysium of TA and BB muscles at 4 months old and 19 months old, and found that both were significantly thicker in the compared with their animals, but not in the and not in the BB also increased in thickness (Fig. 1H). Table 1 Quantification of SEM images for endomysium and Ac-Gly-BoroPro perimysium connective content of 4-month-old and 19-month-old and mice (Fig. 2ACD). Immunohistology revealed relatively uniform laminin expression around 4-month-old BB muscle fibres (Fig. 2A). In contrast, laminin expression was at lower levels and varied in thickness, often showing discontinuity in BB fibres from 4-month-old muscle (Fig. 2D). We evaluated the distribution of the non-fibrillar type IV collagen, another major component of the basal lamina. Immunohistology showed that the expression level of collagen IV was high and uniform in 4-month-old in contrast to the low expression profile in age-matched mice (Fig. 2F,H). Open in a separate window Fig. 2 Myostatin deletion changes expression of extracellular matrix proteins in the mouse muscles. Representative images of mid-belly transverse section of BB muscle of 4-month-old and 19-month-old and showed an increase in the thickness and density of laminin (arrow) compared with 4-month-old muscle between 4 and 19 months (arrows). (G, H) In contrast, collagen thickness remained constant in muscle muscle between 4 and 19 months (arrows). (K, L) In contrast, collagen thickness failed to increase in thickness with time in muscle (arrows). (O, P) In contrast, abundance of collagen III decreased over time in in muscle (arrows). (S, T) MMP-9 Ac-Gly-BoroPro abundance decreased in muscle, respectively. Note Ac-Gly-BoroPro thinner layer of collagen between MHCIIB-expressing fibres (purple arrowheads) compared with thicker collagen layer adjacent to non-MHCIIB fibres (yellow arrowheads). (XCZ) Collagen IV (green), MHCIIB (red) and dual stain for collagen IV (green) and MHCIIB (red) in (Fig. 2I,K). Furthermore, the abundance of collagen I increased between 4 and 19 months in but not in BB (Fig. 2M,N). Furthermore, we observed that the ENO2 concentration of collagen III was higher around small fibres compared with the large fibres in BB (Fig. 2M,N). On the other hand, 4-month-old compared with age-matched and (Fig. 3a, left). Furthermore, quantitative analysis showed that there was significantly more of each protein at 4 months in compared with muscle. In addition, consistent with immunohistochemistry data, and (red) and (= 0.001 and 0.001), respectively. A significant increase in protein levels in 19-month-old compared with 4-month-old was detected for collagen I and III ( 0.05 and = 0.08), respectively. Significant interaction (age vs. genotype) indicates the difference between both genotypes with ageing for collagen I and III ( 0.05 and = 0.001), respectively. (d) Quantification of the.