Enzyme Histochemistry: Identification of Fast vs Slow
Twitch Muscle fibers in Rat Tissue
Skeletal muscle is made up of bundles of individual muscle fibers called myocytes. Each
myocyte contains many myofibrils, which are strands of proteins (actin and myosin) that can
grab on to each other and pull. This shortens the muscle and causes muscle contraction.
It is generally accepted that muscle fiber types can be broken down into two main types: slow
twitch (Type I) muscle fibers and fast twitch (Type II) muscle fibers. Fast twitch fibers can be
further categorized into Type IIa and Type IIb fibers.
Slow twitch muscles are more efficient at using oxygen to generate more ATP for
continuous, extended muscle contractions over a long time. They fire more slowly than fast
twitch fibers and can go for a long time before they fatigue. Therefore, slow twitch fibers are
great at helping athletes run marathons and bicycle for hours.
Since fast twitch muscle fibers use anaerobic metabolism to create fuel, they are much
better at generating short bursts of strength or speed than slow muscles. However, they fatigue
more quickly. Fast twitch fibers generally produce the same amount of force per contraction as
slow muscles, but they get their name because they are able to fire more rapidly. Having more
fast twitch fibers can be an asset to a sprinter since s/he needs to quickly generate a lot of force.
Histochemical methods can be used to classify muscle fibers into fast or slow, oxidative
or non-oxidative, and glycolytic or non-glycolytic. "Histochemical" [histo = tissue] implies that
the chemical reaction is occurring in the tissue itself, rather than in a test tube or other reaction
vessel. Histochemical methods rely on the fact that enzymes located in thin (6-8 µm) frozen
sections of muscle fibers can be chemically reacted with certain products in order to visualize the
activity of the enzyme.
The basic requirement for a histochemical assay is similar, at least in principle, to the
requirement for any biochemical assay. First, a substrate is provided for the enzyme to be
studied. Second, an energy source is provided that allows the enzyme to utilize the substrate.
Finally, a reaction product is linked to another product that forms a precipitate so it can be
visualized microscopically.
There are three histochemical assays typically used to determine muscle fiber types.
These three assays are the myosin ATPase (mATPase) assay, the succinate dehydrogenase
(SDH) assay, and the -glycerophosphate dehydrogenase (αGPD) assay. The mATPase assay is
used to distinguish between fast- and slow-twitch muscle fibers. Since ATP is hydrolyzed during
force generation, the level of ATPase activity can be correlated to contraction speed. The a-GDP
assay is used to identify glycolytic potential. The function of -GDP is to shuttle NADH into
mitochondria where the electrons it carries can be used to make ATP. Cytoplasmic NADH is
produced during glycolysis and therefore is an indirect measure of glycolytic activity.
The histochemical assay for SDH is used to distinguish between oxidative and nonoxidative (actually, “less” oxidative) fibers. Fibers with a high oxidative capacity generate ATP
via oxidative phosphorylation in the mitochondria. It follows that muscle cells that contain more
mitochondria will have a higher oxidative capacity and therefore will have a higher contraction
speed (a fast fiber). Therefore SDH levels can be an indicator if a muscle fiber is a fast twitch or
a slow twitch fiber.
In this lab, you will dissect muscle tissue from a rat. This muscle tissue will be frozen in
isopentane cooled to -1400C followed by sectioning with a cryostat. The frozen sections will then
be stained for SDH activity.
1
SDH is located in the inner membrane of the mitochondrion, bound to the cristae. SDH is
responsible for oxidizing succinate to fumarate in the Kreb’s Cycle. As this reaction proceeds,
succinate is oxidized, and the reduced form of NADH is produced. Succinate is therefore the
substrate, NADH is the reaction product (actually, a different electron acceptor is used for
practical reasons), and SDH is the enzyme. The electron acceptor is chemically reacted with nitro
blue tetrazolium (NBT), a purple salt, to visualize the reaction, and this results in a speckled
pattern of the mitochondria (Figure 1), proportional to the number of mitochondria and the SDH
activity within them. Similar to the ATPase assay, the more SDH (and therefore mitochondria) a
fiber contains, the greater the intensity of the stain. Oxidative fibers have a relatively dense,
purple speckled appearance, while non-oxidative fibers have only scattered purple speckles.
Therefore, this histochemical assay reflects the relative oxidative potential of muscle fibers.
Purpose: The purpose of the this lab is to use a histochemical stain for a SDH to identify fast
and slow muscle fibers in rat thigh muscle, determine the percentage of these fibers in the tissue
and the average diameter of each fiber type.
Fig. 1 Frozen section of skeletal muscle stained for SDH showing fast fibers (dark) and slow fibers
(light). Retrieved from the World Wide Web at http://muscle.ucsd.edu/musintro/histochem.shtml
on January 9, 2008
Procedure:
1. Freezing muscle specimens
a. Dissect out muscle specimen. Suggestions would be abdominal or leg muscle.
b. Rinse tissue in ice cold PBS to remove blood. Leave in PBS until ready to freeze.
c. Place the plastic container into a larger plastic container. Fill the smaller container ½ full
with isopentane (2-methylbutane). Slowly add small chips of dry ice to the isopentane.
Watch out for boiling over. Keep adding dry ice until the isopentane stops boiling.
d. Using forceps drop your tissue into the isopentane and allow to remain for about 15
sections. Then remove the specimen and place on the Saran wrap on dry ice. Don’t let the
sample thaw.
2. Mounting specimen for sectioning
a. Make sure the cryostat is set to -20oC.
b. Transport the specimen to the cryostat on dry ice.
c. Mount the specimen on a disk using freezing medium.
2
d.
e.
f.
g.
Allow the specimen to sit in the cryostat for 10 minutes to warm up.
Section specimen at 8 m.
Sections should be collected toward the bottom of the slide.
Slides can be kept in a slide box until ready for staining.
3. Staining slides for SDH activity.
a. Incubate sections for 30 minutes at 37oC in incubation medium placed in a Coplin jar.
b. Rinse section in physiological saline. This can be done by dipping the slides several times
in a Coplin jar containing the saline.
c. Fix sections in 10% formalin-saline solution (Coplin jar) for 3-5 minutes.
d. Rinse in 15% alcohol for 5 minutes (Coplin jar).
e. Mount with an aqueous mounting medium and let sit for 2-3 minutes.
f. Seal edges of cover slip with clear nail polish and let dry.
g. View with a microscope.
0.2 M. Sodium phosphate monobasic
2.78 g/100 ml
0.2 M Sodium phosphate dibasic
5.37g/100ml
0.2 M Phosphate Buffer, pH 7.6
13 ml 0.2 M sodium phosphate monobasic
87 ml 0.2 M sodium phosphate dibasic
Check pH and adjust to 7.6 using 1N NaOH or 1N HCL. Store in the refrigerator.
0.2 M Sodium succinate solution
2.70 g Sodium succinate (NaOCOCH2CH2COONa*6H20
50 ml dH20
Prepare fresh
NBT solution
0.1 g NBT (Sigma # N-6876)
50 mls dH20
Make up only 50 ml at a time and store in the refrigerator.
Incubation medium
Just before use, mix: 5 ml 0.2 M phosphate buffer
5 ml sodium succinate solution
5 ml NBT solution
5 ml dH2O
Physiological Saline
8.5 g NaCl
1 L dH2O
Store at room temperature
3
10% Formalin-saline solution
45 ml physiological saline
5 ml 40% formaldehyde
Prepare fresh each time
15% alcohol
15 ml ETOH
85 ml dH2O
Buffered Glycerol mounting medium
Either 0.1M Phosphate buffer (pH 7.4): 10 ml or 0.1M TRIS buffer (pH 9.0): 10 ml
Anti-fading agent: Either p-Phenylenediamine hydrochloride: 100 mg or n-propyl gallate: 500 mg
Glycerol: 90 ml
Keeps for at least 3 months, probably much longer, in darkness (which protects the anti-fade agent)
at -20C. The working bottle is kept at 4C, for a week or two.
Data analysis:
1. Determine the percentage of fast vs slow fibers.
2. Determine the average diameter of fast and slow fibers.
3. Take a picture of your preparation.
Lab report:
1. Write an abstract and a results section of your research including all necessary data properly
presented.
2. Include a picture of your slide that includes a size marker.
3. Answer the following questions;
a. For each of the following indicate its purpose.
 NBT
 Succinate
 Phosphate buffer
 Formalin-saline
b. Why are some muscle fibers stained darker than others?
c. Explain how this stain works to label mitochondria.
d. What is the difference between fast and slow fibers?
e. Why do muscle cells have these two different types of fibers?
f. If you are an endurance runner, is it better have more fast fibers or slow fibers? Explain.
This protocol was obtained at
http://www.ihcworld.com/_protocols/histology/aqueous_mounting_medium.htm.
Retrieved from the World Wide Web on January 10, 2008
This lab was adapted from Histotechnology: A Self-Instructional Text (1997)
by Freida L Carson, ASCP Press, p. 265-267
4