Noq7.5.4d

Excised gastrocnemius and soleus tissues were fixed with 4% paraformaldehyde and embedded in paraffin. Tissue samples were cut at a thickness of 6 µm and the sections were stained with hematoxylin-eosin (HE). The mean CSA of fibers was determined using the Image J software (National Institute of Health). Immunohistochemical staining was performed with a monoclonal antibody against skeletal slow (NOQ7.5.4D, Sigma) and fast myosin (MY-32, Sigma).

TA, soleus and gastrocnemius muscles were embedded in cryomatrix and quickly frozen in isopentane cooled with liquid nitrogen. Cryostat sections (10 µm) were fixed in 4% PFA, washed in PBS, permeabilized with 0.1% Triton X-100 and left for 1 hour in blocking solution (1× PBS, 1.5% goat serum, 0.1% Triton X-100). Rabbit poly-clonal antibodies directed against Laminin (Z0097, Dako) (1/100 dilution), and monoclonal antibodies against MYH7 (NOQ7.5.4D, Sigma) (1/1000 dilution), MYH2 (SC-71, Developmental Studies Hybridoma Bank) (1/20 dilution) and against MYH4 (BF-F3, Developmental Studies Hybridoma Bank) (1/20 dilution) were applied overnight at 4°C to the treated sections. The next day, after three washes with 1× PBS containing 0.05% Tween-20, cryosections were incubated for 1 h with appropriate fluorescent secondary antibodies (Alexa Fluor 488 goat anti-rabbit IgG 1/1000 dilution, Alexa Fluor 594 goat anti-mouse IgG 1/1000 dilution, Invitrogen). After three washes with 1× PBS containing 0.05% Tween 20, samples were mounted in Vectashield mounting medium.

Antibodies to peptides in hSOD1 (corresponding to aas 4–20, 24–39, 41–49, 43–57, 48–57, 57–72, 80–96, 94–102, 100–115, 115–121, 131–153, 132–140 and 144–153) that were coupled to keyhole limpet hemocyanin were raised in rabbits and affinity purified with immobilized peptides as described (Ra-Abs) [15 (link), 20 (link)]. Antibodies raised to the C-terminal end in hSOD1^G127X, aas 111–127GQRWK and 123–127 GQRWK, were prepared in the same way. A chicken antibody (Ch-Ab) against aas 131–153 was also raised and affinity purified. A monoclonal mouse antibody (Mo-Ab) against aas 131–153 was produced using standard hybridoma technique. For muscle immunohistochemistry, an antibody against slow myosin was used (clone NOQ 7,5,4D; Cat no M8421, Sigma). For motor neuron staining and counting, an antibody against choline acetyltransferase (ChAT) (abcam EPR 16590) was used.