Stains & CD markers

Editorial Board Member: Christian M. Schürch, M.D., Ph.D.
Deputy Editor-in-Chief: Chunyu Cai, M.D., Ph.D.
Moiz Vora, M.D.

Last author update: 28 July 2022
Last staff update: 28 July 2022

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PubMed Search: NADH

Moiz Vora, M.D.
Page views in 2022: 1,132
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Cite this page: Vora M. NADH. website. Accessed June 2nd, 2023.
Definition / general
  • Nicotinadmide adenine dinucleotide (NADH) is a coenzyme that facilitates substrate reducing reactions associated with glycolysis, oxidative phosphorylation and fermentation
  • NADH-TR stain protocol utilizes enzymatic activity to release hydrogen from NADH, producing a purple-blue formazan pigment that marks the reaction site
Essential features
  • NADH enzyme histochemical stain reveals myofibrillar architecture, mitochondria and target fibers; it helps differentiate type I (oxidative) and type II (nonoxidative) fibers
  • This technique demonstrates patterns of myofiber injury that are characteristic of congenital and mitochondrial myopathies and specific muscular dystrophies
  • This stain protocol requires the use of snap frozen muscle biopsies
  • Nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR)
  • Nicotinamide adenine dinucleotide diaphorase activity
  • Mitochondria within sarcoplasmic network of striated muscle
Uses by pathologists
  • Reveals architectural changes in the muscle, e.g., central cores, whorled, lobulated and moth eaten fibers; these changes assist in categorizing patterns of myofibril injury (J Histotechnol 2008;31:101, ScienceDirect: Tetrazolium Reductase [Accessed 2 February 2022])
  • NADH diaphorase activity, in combination with myosin ATPase activity at pH 9.4, allows fibers to be classified into 3 categories:
    1. Slow twitch high oxidative (ST)
    2. Fast twitch high oxidative (FTH)
    3. Fast twitch low oxidative (FT)
Microscopic (histologic) description
  • Speckled pattern within myofibers (J Histotechnol 2008;31:101)
  • Intensity is proportional to number of mitochondria and NADH activity
    • Type I oxidative fibers (dark, dense purple appearance)
    • Type II nonoxidative fibers (light, scattered purple speckles)
Microscopic (histologic) images

Contributed by Chunyu Cai, M.D., Ph.D.

Normal NADH

Myopathy with lobulated fibers

Central core myopathy

Multiminicore myopathy

Nemaline rod myopathy

Target fibers

Myopathy with tubular aggregates

Necklace fibers

Ragged blue fibers

Dermatomyositis perifascicular atrophy

Myofibrillar myopathy

Positive staining - normal
Positive staining - disease
Negative staining
Board review style question #1

In which type of myopathy is this characteristic pattern of injury typically seen by NADH-TR histochemistry?

  1. Central core disease (type I fibers with central cores)
  2. Debranching enzyme deficiency (ring fibers)
  3. Dermatomyositis (perifasicular atrophy)
  4. Mitochondrial myopathy (ragged blue fibers)
  5. Tubular aggregate myopathy (tubular aggregates)
Board review style answer #1
D. Mitochondrial myopathy (ragged blue fibers)

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Reference: NADH
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