VitroView™ COX-SDH Double Histochemistry Stain Kit (For 50-100 Slides)

$495.00

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SKU: VB-3022 Category:

Description

Analyzing respiratory enzyme activity is a powerful method for identifying mitochondrial dysfunction. The COX/SDH Double Stain Kit enables the visualization of cytochrome c oxidase (COX) and succinate dehydrogenase (SDH) activity, providing critical insights into mitochondrial pathology. COX, essential for mitochondrial function, has three subunits encoded by mitochondrial DNA (mtDNA), making it susceptible to mtDNA mutations. In contrast, SDH is entirely encoded by nuclear DNA and remains unaffected by mtDNA impairments. This dual staining technique effectively highlights COX-negative fibers, which may appear SDH-positive and ragged red fibers—hallmarks of mitochondrial myopathies. Additionally, intra-fiber mosaicism (a mix of COX-deficient and COX-positive mitochondria within the same fiber) is clearly visualized. The COX/SDH stain is a valuable tool for studying mitochondrial diseases, aging-related disorders, and conditions like Mitochondrial Encephalopathy, Lactic Acidosis, and Stroke-like Episodes (MELAS), where COX activity may still be preserved.

Kit Components

SKU# Reagent Size
VB-3022-1 COX A Solution 1ml×5
VB-3022-2 COX B Solution 1ml×5
VB-3022-3 Succinate  Solution 0.3ml×5
VB-3022-4 Yellow SDH Incubation Medium 1.5 ml×5
VB-3022-5 COX Inhibitor Solution 1ml×2
VB-3022-6 SDH Inhibitor Solution 1ml×2

Storage

Store at -20°C

Protocol

  1. Tissue preparation for cryosectioning
  • Sacrifice the animal by either cervical dislocation or decapitation, in accordance with available ethical permit.
  • Quickly collect tissues of interest without fixation, and rapidly freeze on dry ice (tissues may require freezing in isopentane) or propane chilled with liquid nitrogen to obtain optimal morphology).
  • Store tissues in aluminum foil at -80 °C until ready to section.
  • Embed frozen tissue in preparation for cryosectioning.
  • Collect 10-14μm cryostat sections. Thaw sections onto slides at room temperature for 2-5 minutes, and store slides without cover-slipping at -20 °C until ready to use.
  1. Prepare COX incubation solution: Defrost one COX A Solution (VB-3022-1) and one COX B Solution (VB-3022-2) vial. One vial of COX A solution are mixed with 1 vial of COX B solution.
  2. Immediately put 80~200ul of COX incubation solution onto frozen sectioned slides in humidity chamber and incubate in dark for two hours at room temperature.
  3. Check staining and replace for longer if required
  4. Rinse slides in distilled water
  5. Freshly prepare SDH incubation medium: Defrost a vial (0.3ml) of Succinate Solution (VB-3022-3) and a vial of Yellow SDH Incubation Medium (1.5 ml) (VB-3022-4); Add solution succinate solution to Yellow SDH Incubation Medium prior to use and mix well.
  6. Add 80~200ul of SDH incubation medium onto slide and incubate at 37 ͦ C for 1-2 hour.
  7. Rinse slides in distilled water.
  8. Rinse in water, dehydrate, clear and mount.

Appropriate specificity controls

  1. For specificity controls for COX activity, repeat “COX histochemistry” steps, and add 100µl of COX Inhibitor Solution (VB-2022-5), a terminal respiratory chain inhibitor.
  2. For specificity controls for SDH activity, repeat “SDH histochemistry” steps with the removal of sodium succinate and the addition of 100µl of SDH Inhibitor Solution (VB-2022-6), a competitive inhibitor of SDH.

Results

Cytochrome Oxidase positive mitochondria——- Brown
Cytochrome Oxidase negative mitochondria——- Blue

More Images

COX Stain Only on mouse muscle section, Cox stained at room temperature for 60 minutes, 20x

 

SDH Stain Only on mouse muscle section, SDH stained at 37ºC for 40 minutes, 20x

 

COX/SDH Double Stain on mouse muscle section, Cox stained at room temperature for 60 minutes, SDH stained at 37ºC for 40 minutes, 20x

 

COX/SDH Double Stain with Cox inhibitor on mouse muscle section, Cox stained at room temperature for 120 minutes, SDH stained at 37ºC for 40 minutes, 20x

 

COX/SDH Double Stain with SDH inhibitor on mouse muscle section, Cox stained at room temperature for 60 minutes, SDH stained at 37ºC for 40 minutes, 20x

 

References

  1. Ross, J.M. Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry. J. Vis. Exp. (57), e3266, DOI : 10.3791/3266 (2011).
  2. Seligman etal (1968) J Cell Biol 38:1-14.
  3. Loughlin M. (1993). Muscle biopsy. A laboratory investigation. Butterworth-Heinemann p.38-39.
  4. Sheehan D, Hrapchak B. (1987). Histotechnology, 2nd Ed. Batelle Press, Columbus p306-307

Recent Publications Using VB-3022

  1. Dombrecht D,  Daele  UV, Asbroeck  BV, David R. Schieffelers DR, Guns PJ,Breda EV (2023). Skeletal muscle wasting after burn is regulated by a decrease in anabolic signaling in the early flow phase. BurnsVolume; 49 ( 7): 1574-1584
  2. Dang X, Zhang L, Franco S, Dorn II GW (2023). Activating mitofusins interrupts mitochondrial degeneration and delays disease progression in SOD1 mutant amyotrophic lateral sclerosis. Human Molecular Genetics; 32 (7): 1208–1222
  3. Mosteiro L, Nguyen TTT, Hankeova S,  Alvarez-Sierra D, Reichelt M,  Vandriel SM, Lai Z, Choudhury FK,  Sangaraju D,  Kamath DM, Scherl A, Pujol-Borrell R, Robert Piskol R & Christian W. Siebel (2023). Notch signaling in thyrocytes is essential for adult thyroid function and mammalian homeostasis. Nature Metabolism; 5: 2094–2110

Note

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

Precautions

Handle with care. Avoid contact with eyes, skin and clothing. Do not ingest. Wear gloves.

User Manual and Material Safety Data Sheet (MSDS)  (PDF)

VB-3022 User manual

VB-3022 MSDS

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