Dear Damian,

please read the following article:

http://www.pnas.org/content/99/12/8418.full

and this part of the above article:

xxxxx

The predominant COX product of the adult zebrafish is PGE2. Although its tissue source is unknown, it is the most abundant COX product of microvascular endothelium in humans (28). PGI2, the predominant product of macrovascular endothelium in humans, was a minor product of whole zebrafish. PGE2 plays an important role in angiogenesis (29), regulation of blood pressure (30), and hemostasis (31) in mice. Marked inhibition of PGE2 synthesis by both nonselective and COX-2-selective inhibitors suggests that COX-2 is the major source of zebrafish PGE2. Indeed, constitutive expression of the zCOX-2 transcript was apparent in blood vessels and the intestine, likely sources of PGE2, in adult fish.

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I worked with vital staining of zebrafishes (eggs, young fish and adults) several years ago but not with DAB staining of tissues of them. DAB stainig (via formation per oxydation of a brown "phenazine polymer" is sometimes very curious (overstaining a.s.o...).

I am interested about your future result with the DAB protocol.

Good luck

JRG

Dear Jean,

there is COX acronym confusion here. The article your referred to concerns cyclooxygenase (COX), also known as prostaglandin-endoperoxide synthase (PTGS). I am interested in Cytochrome C oxidase, which also has the acronym COX. I will post an update on Cytochrome C oxidase staining.

Regards, Damian

Update.

I found a poorly cited paper that exactly describes a cytochrome C oxidase staining protocol for zebrafish via DAB histochemistry.

Van Raamsdonk, W., Van Den Bogert, C., Smit-Onel, M. J., Muijsers, A. O. and Diegenbach, P. C. (1994). Combined quantitative immuno- and enzyme cytochemistry of cytochrome c oxidase in sections of neural tissue and cultured cells. Acta Histochemica 96, 19-32.

The protocol uses COX derived from horse heart and an incubation temperature of 40°C. So it appears the be the same technique used for mammalian tissue:

Tanji, K. (2012). Morphological Assessment of Mitochondrial Respiratory Chain Function on Tissue Sections. Methods in Molecular Biology 837, 181-194.

Ross, J. M. (2011). Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry. Journal of Visualized Experiments, vol. 57, pp. e3266.

Hi Damian,

I have used cytochrome oxidase histochemistry in goldfish for many years. The difficulty regarding applying the technique to fish is achieving a high quality perfusion fixation via the conus arteriosus. Typical perfusion needles can be used in larger fish, but in small fish such as goldfish or zebrafish you would need to set up a perfusion apparatus involving a micropipette hooked up with tubing with syringe and a micro-drive to advance the pipette into the conus under the guidance of a stereomicroscope. You will also need to cut the gills to allow the blood to flow out as you advance the fixative. If you use immersion fixation you would face two problems: 1. Blood cells will react with the DAB, and 2. You will get uneven/unreliable staining because the histochemical reaction is affected by the quality of tissue fixation and the type of fixative that is used. I can refer you to several research papers by Kageyama, GH and RL Meyer on the topic of cytochrome oxidase histochemistry in goldfish. This will not be easy with zebrafish because of their size. It would be challenging even for me, and I have had many years of experience with various species of fish.

My best,

Glenn

Could someone advise me on the correct equation to use to calculate Cytochrome Oxidase activity in tissue homogenates in a spectrophotometric assay where a known amount of Cytochrome C is added?

I found this:

(Delta-Abs/ EC)/ [g of tissue]

where:

- Delta Abs = difference in absorbance at 550 nm in 1 min

- EC = extinction coefficient of reduced Cytochrome C - extinction coefficient

oxidized Cytochrome C (both at 550 nm) = 19.6/ mM cm

I am having a hard time though to understand because the EC is given in different values with different orders of magnitude...(e.g. Gonzalez-Lima 1998 - book chapter: 19.6 micromolar, Hess and Pope 1953 - 19.6 millimolar).

Is it correct that EC = 19.6 / milliMolar * cm? or is it 19.6 micromolar??

CO activity is defined as 1 unit oxidizes 1 microMole of reduced Cyt C in 1 min. So If I use EC = 19.6 milliMol it means that I have to divide my value by 1000 to obtain the enzyme units number, is that correct?