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

Summary

Here we demonstrate the most efficient methods for freezing, embedding, cryosectioning, and staining of muscle biopsies to avoid freezing artifacts.

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.

Introduction

Examination of muscle histology plays a critical role in the understanding of different types of neuromuscular disorders^1-4. When combined with other techniques, it allows researchers to get a better idea of the manifestation and progression of pathology and can also be essential to evaluate the efficacy of a therapeutic intervention^5-10. However, to be accurate, the tissues need to be preserved in the proper manner so as to preserve the physiological state immediately before excision. This requires great care during removal, preservation, sectioning, and staining.

Tissues can be prepared in two different ways for ligh....

Protocol

1. Tissue Harvest and Freezing of Muscle Tissues

1. Euthanize the mouse with an overdose of isoflurane (2-chloro-2-(difluoromethoxy)-1,1,1-trifluoro-ethane). Perform this step in a fume hood and use a secondary containment method such as a desiccator or a bell jar containing a cotton swab soaked in isoflurane.
2. Confirm death by firmly squeezing footpad. Use a secondary method of euthanasia such as cervical dislocation.
3. Wet the fur with 70% alcohol to reduce the sticking of loose hair strands on the muscles. Peel the skin off the limb using a fine forceps such as #5.
4. Carefully separate the muscle of interest (tibialis anterior (TA)....

Results

The set-up for harvesting and freezing of tissues can be seen in Figure 1. Excised tissues should be stored on a gauze soaked in PBS to avoid drying (Figure 1A). When tissues are selected to be used for histological analysis they should be dipped in OCT and laid out on cryo mold in the proper orientation and as close to physiological length as possible to ensure accurate morphology (Figure 1B). Slurry of isopentane suitable for tissue freezing can be achieved by cooling .......

Discussion

Histology and immunohistochemistry have been used as key tools in understanding the manifestation and progression of pathology in various neuromuscular disorders. Classically, muscle biopsies were first fixed in formaldehyde and then embedded with paraffin wax. While formaldehyde fixation preserves tissue architecture and allows tissue storage and transport at RT, it also inactivates enzymes and crosslinks proteins necessitating further steps to achieve accurate immunostaining. This problem is eliminated with the use of .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cryostat	Leica CM 1850	Leica Microsystems Inc.	Buffalo Grove, IL 60089 U.S.A., office phone 1-800-248-0123 Alternative: Richard Allen Scientific
Microscope	Nikon Eclipse 50i	Nikon Instruments Inc.	1300 Walt Whitman Road, Melville, N.Y. 11747-3064 U.S.A., Phone: 1-631-547-8500 Alternative: Olympus
Image analysis software	Nikon Imaging Software Basic Research	Nikon Instruments Inc.	1300 Walt Whitman Road, Melville, N.Y. 11747-3064, U.S.A., Phone: 1-631-547-8500 Alternatives: Image J, Metamorph
Isoflurane	14043-220-05	JD Medical Dist. Co., Inc.	1923 West Peoria Avenue, Phoenix, AZ 85029 U.S.A.
OCT	4583	Sakura Inc.	Torrence, CA 90501 U.S.A. Alternative source: Leica Microsystem
Freeze It	23-022524	Thermofisher	790 Memorial Dr, Cambridge, MA, 02139 U.S.A. Alternative source: VWR Scientific
Disposable tissue mold	22-363-554	Thermofisher	790 Memorial Dr, Cambridge, MA 02139 U.S.A. Alternative source: VWR Scientific
Colorfrost plus slides	9991001	Thermofisher	790 Memorial Dr, Cambridge, MA 02139 U.S.A. Alternative source: VWR Scientific
Acetone	650501-4L	Sigma-Aldrich	3050 Spruce Street, St. Louis, MO 63103 U.S.A. Alternative source: VWR Scientific
Ethyl alcohol	HC-1300-1GL	Thermofisher	790 Memorial Dr, Cambridge, MA 02139 U.S.A. Alternative source: VWR Scientific
2-methyl butane	M 32631-SL	Sigma-Aldrich	3050 Spruce Street, St. Louis, MO 63103 U.S.A. Alternative source: VWR Scientific
Xylene	HC-7001GA	Thermofisher	790 Memorial Dr, Cambridge, MA 02139 U.S.A. Alternative source: VWR Scientific
Cytoseal 280	8311-4	Thermofisher	790 Memorial Dr, Cambridge, MA 02139 U.S.A. Alternative source: VWR Scientific
Hematoxylin Gill #3	CS402-1D	Thermofisher	790 Memorial Dr, Cambridge, MA 02139 U.S.A. Alternative source: VWR Scientific
Eosin	6766007	Thermofisher	790 Memorial Dr, Cambridge, MA 02139 U.S.A. Alternative source: VWR Scientific