Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis

Abstract

Histological evaluation of muscle biopsies has served as an indispensable tool in the understanding of the development and progression of pathology of neuromuscular disorders. However, in order to do so, proper care needs to be taken when excising and preserving tissues to achieve optimal staining. One method of tissue preservation involves fixing tissues in formaldehyde and then embedding them with paraffin wax. This method preserves morphology well and allows for long-term storage at RT but is cumbersome and requires handling of toxic chemicals. Further, formaldehyde fixation results in antigen cross-linking, which necessitates antigen retrieval protocols for effective immunostaining. On the contrary, frozen sectioning does not require fixation and thus retains biological antigen conformation. This method also provides a distinct advantage in quick turn around time, making it especially useful in situations needing quick histological evaluation like intraoperative surgical biopsies. Here we describe the most effective method of preparing muscle biopsies for visualization with different histological and immunological stains. Video LinkThe video component of this article can be found at

UCHL1 regulates oxidative activity in skeletal muscle

“…Soleus, EDL, and gastrocnemius are primarily composed of slow oxidative fibers, fast glycolytic fibers, and mixed muscle fibers, respectively. For tissue staining, gastrocnemius muscles were isolated and snap frozen in pre-chilled 2-methylbutane for ~20 seconds [ 21 ] and transferred to -70°C until sectioning. Mid-belly region of muscles was sectioned into 10 μm thickness and adhered on the slides.…”

A Multi-hole Cryovial Eliminates Freezing Artifacts when Muscle Tissues are Directly Immersed in Liquid Nitrogen

“…However, the water content of muscles is greater than 75% in pigs, rabbits, mice, and humans, regardless of the position (i.e., back, abdomen, or hindlimb) 7 . Such high moisture content in muscles causes a commonly encountered issue -freezing artifacts -during the preparation of cryosections, as previously described 8,9 . In most cases, it is almost impossible to appropriately freeze muscle tissues in a slaughterhouse production line, according to our experience.…”

“…Importantly, this method can be used efficiently in a high-throughput environment because it is easy to perform and saves time (Figure 4). Nowadays, a well-established freezing method is to submerge muscle tissues in a slurry of isopentane without contact with OCT mounting medium, as previously described 8,9 ( Figure 3F). In this procedure, it takes approximate 30 min for a solid-liquid slurry state of 240 mL of isopentane to be achieved by pre-cooling the isopentane in liquid nitrogen and stirring until small white precipitates appear at the bottom (Figure 4).…”

Current Approaches in Immunoassay Methods Focus on Skeletal Muscle Proteins

“…Following freezing and until sectioning, tissue must be stored at ultra-low temperature (−80°C). The tissue architecture undergoes damage due to freeze-thaw [22]. Also, longer storage tissue sections lead to cracking as well as decrease in intensity signal levels.…”

“…The usefulness of the method lies in the identification of the primary protein abnormalities in recessive diseases [33] and secondary reduction of interconnected proteins. For example, in the primary reduction of dystrophin, protein involved in Duchenne/Becker muscular dystrophy and secondary reduction of sarcoglycans [22] and of cytosolic calpain 3 [25] was reported. The reduction of dysferlin also is accompanied by the reduction of calpain 3.…”