Isolation of Mouse Periocular Tissue for Histological and Immunostaining Analyses of the Extraocular Muscles and Their Satellite Cells

Abstract

The extraocular muscles (EOMs) comprise a group of highly specialized skeletal muscles controlling eye movements. Although a number of unique features of EOMs including their sparing in Duchenne muscular dystrophy have drawn a continuous interest, knowledge about these hard to reach muscles is still limited. The goal of this chapter is to provide detailed methods for the isolation and histological analysis of mouse EOMs. We first introduce in brief the basic anatomy and established nomenclature of the extraocular primary and accessory muscles. We then provide a detailed description with step-by-step images of our procedure for isolating (and subsequently cryosectioning) EOMs while preserving the integrity of their original structural organization. Next, we present several useful histological protocols frequently used by us, including: (1) a method for highlighting the general organization of periocular tissue, using the MyoD(Cre) × R26(mTmG) reporter mouse that elegantly distinguishes muscle (MyoD(Cre)-driven GFP(+)) from the non-myogenic constituents (Tomato(+)); (2) analysis by H&E staining, allowing for example, detection of the pathological features of the dystrophin-null phenotype in affected limb and diaphragm muscles that are absent in EOMs; (3) detection of the myogenic progenitors (i.e., satellite cells) in their native position underneath the myofiber basal lamina using Pax7/laminin double immunostaining. The EOM tissue harvesting procedure described here can also be adapted for isolating and studying satellite cells and other cell types. Overall, the methods described in this chapter should provide investigators the necessary tools for entering the EOM research field and contribute to a better understanding of this highly specialized muscle group and its complex micro-anatomy.

Mass Spectrometric Profiling of Extraocular Muscle and Proteomic Adaptations in the mdx-4cv Model of Duchenne Muscular Dystrophy

“…In order to evaluate the expression of glyceraldehyde-3-phosphate dehydrogenase in wild type versus mdx-4cv EOM muscle, immunofluorescence microscopy was carried out by standardized methodology in combination with histological staining [ 49 ]. Freshly dissected skeletal muscle specimens from mice were quick-frozen in liquid nitrogen-cooled isopentane and 10 μm sections were cut in a cryostat [ 50 ]. Tissue sections were fixed in a 1:1 ( v/v ) mixture of methanol and acetone for 10 min at room temperature and then blocked with 1:20 diluted normal goat serum for 30 min at room temperature.…”