Perlecan

Immunofluorescence and haematoxylin and eosin staining were carried out
as described previously^{9 (link)}. All
images are representative from larger analysis of at least three animals per
group, per condition. Fibre size diameter (minimal Feret’s diameter) was
assessed using ImageJ software (version 1.45s), based on β-DG
immunofluorescence. Necrosis was assessed using anti-mouse IgG fluorescent
antibody (detects immune-cell infiltration of degenerating fibres). qRT-PCR for
mouse Dag1 and associated glycosyltransferases was performed
using a BioRad MyIQ system, with protocols and primer sets previously
described^{32 (link)}. The
following primer sets were also used: Ispd1 forward
5′-TGGTGTGGATTAGGGGGTTA-3′, reverse
5′-TGGCTGCACTTTGTCCTAAA-3′; Tmem5 forward
5′-GAGAACAGTGGCAGCCTCA-3′, reverse
5′-CAAAGGAGCAGGCCTCATAG-3′; Sgk196 forward
5′-GCTGTCCTGTGAAGAGCTGA-3′, reverse
5′-GGGAGAGAGCGACTTTGTGT-3′; Ignt1 forward
5′-ACATTTGACGAACGCTTTC-3′, reverse
5′-CCTCCTTTTGGGGATGGAAC-3′; Itga7 forward
5′-TTGCTGTTAGCCACGATCAG-3′, reverse
5′-CGCCAGAGAAGAAGAGTTGC-3′; Col6a forward
5′-CTCTCCTGGTTCACCCATGT-3′, reverse
5′-CCCGACTCTACCGAGATTGA-3′; Lama2 forward
5′-CCAAGAAGGAGGCTGCATAG-3′, reverse
5′-CCAGGTGTTGGGAAGACACT-3′; Lamb1 forward
5′-GTTCGAGGGAACTGCTTCTG-3′, reverse
5′-GTTCAGGCCTTTGGTGTTGT-3′; perlecan(Hspg2)
forward 5′-GAGCGGACTGTACCTTGGTC-3′, reverse
5′-ACCAGTTGCACACAGCTCAC-3′; agrin forward
5′-CAGTGGGGGACCTAGAAACA-3′, reverse
5′-ACCTTTCCAATCCACAGCAC-3′.
Fold changes were calculated using the ΔΔCt method. Failed
reactions, those with Ct values within 1 Ct value of water control, were
excluded. Mouse and human protein samples were obtained by 1% Triton X-100
solubilization of physically disrupted tissue. Glycoprotein enrichment via WGA
agarose beads (Vector Labs) was performed as described previously^{9 (link)}. Densitometry was performed
using LiCor Odyssey Software, V3.0, of triplicate blots. Laminin overlay and
solid-phase assays were performed as previously described^{3 (link),9 (link)}. For analysis of NMJs, Alexa-488-conjugated
α-bungarotoxin was used to stain 4% paraformaldehyde-fixed tibialis
anterior muscle cut into thirds longitudinally. Labelled muscle was cleared
using glycerol and flattened. Maximal-intensity projections were obtained using
an FV1000 Olympus Scanning Confocal laser microscope and
z-stack sections compiled with FluoViewer-1.5 (Olympus). Next,
individual NMJs from each image were assigned a number, cropped and copied onto
a scoring PowerPoint file in a randomized fashion before scoring. Individual
NMJs were scored by three blinded observers, using criteria established
previously^{33 (link)}. NMJs
were scored as ‘fragmented’ if they were comprised of 5 or more
AChR islands, and ‘irregular” if they were abnormally shaped
(either shallow folds and involutions or none at all). NMJs throughout the
muscle were sampled, from 3 animals per group. CAG-Large mice
were analysed for fibre size diameter (minimal Feret’s), fibre
cross-sectional area and density from images acquired using a VS120-S5-FL slide
scanner microscope (Olympus) with VS-ASW (version 2.6). Analysis was carried out
using VS-Desktop software version 2.6.