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Expression of extracellular protein Laminin 9 alpha-4 chain in human skeletal muscle. Indirect immunostaining with HRP immunostain marker. Ob.x40.

I have unsuccessfully searched NCBI -database, JSTOR and other major Biology databases for an answer. This suggests to me that I do not understand what is going on.

1. What are the substrates of above cytochemical reactions for the given immunostaining above?


I need to give a simpler question where I apparently know the reaction exactly, since it is possible that people cannot answer the above question.

Mitochondria in Hep-2 cell line cells. Cytochemical test for mitochondria specific enzyme NAHD dehydrogenase. Ob.x40.

2. What is the substrate of the cytochemical reaction?

My answer:

The reaction of NADH dehydrogenase is:

NADH + H+ + CoQ → NAD+ + CoQH2

Substrate: CoQ

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I'm having a hard time understanding this, it looks like some context is missing. If you're posting homework, please provide the full text of the problem and show what you've tried so far and what exactly you don't understand. –  Mad Scientist Dec 18 '11 at 22:14
    
@MadScientist: Please, see pages 3 and 4 of the file: dl.dropbox.com/u/175564/Practical%20works%202011-2012.pdf –  Masi Dec 18 '11 at 22:49
    
For my benefit can I ask why the in and out are included in the equation? –  Rory M Dec 19 '11 at 10:49
    
@Rory Because I was stupid when copy-pasting the given equation without deleting them, since we do not need them in my opinion. Right? –  Masi Dec 20 '11 at 10:30
    
@Rory I removed 4H from the both sides. Correct now? –  Masi Dec 20 '11 at 10:32

1 Answer 1

up vote 4 down vote accepted

It's hard to understand the question, but in any immunocytochemical staining such as the above, you have two different types of reactions:

  • the antibody binding to the target (in this case, some laminin)
  • the peroxidase-based colorimetric reaction with DAB. DAB (3,3'-diaminobenzidine tetrahydrochloride) is oxidized in the presence of hydrogen peroxide to form a brown precipitate, which becomes the stain.

The fact that is an indirect immunostaining implies that you have a non-conjugated primary antibody against laminin and a secondary antibody against the Fc part of immunoglobulins of the species in which the first antibody was raised. This secondary antibody will be conjugated with HRP (horseradish peroxidase, the enzyme responsible of generating the peroxides that will oxidize the DAB).

For a more complete reference of immunocytochemical stainings, you can read: http://www.ihcworld.com/_books/Dako_Handbook.pdf

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Thank you for your answer! Need to read the book in my Christmas holiday. –  Masi Dec 19 '11 at 5:23

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