Hi all, I performed cytochrome c oxidase staining in muscle cross sections to measure mitochondrial activity based on fiber types. I would like to quantify the intensity of the staining in different fiber types using Image J. Has anyone performed such analysis?
Immunohistochemical staining of cytochrome c oxidase in muscle as the following: Muscle biopsy sections (10 μm) were fixed in 4% paraformaldehyde (PFA) for 10 min at 4 °C before being permeabilised in solutions of Tris-buffered saline Tween-20 pH 8.0 (TBST) then 70% methanol and then 95% methanol with 0.3% H2O2 for 10 min each and 100% methanol for 20 min followed by rehydration. A monoclonal antibody raised against NDUFB8 (a structural component of mitochondrial complex I (complex I-20)) were purchased from Abcam (UK) and used at a concentration of 1:400. The Menapath X-Cell Plus HRP Polymer detection system (A. Menarini Diagnostics, Wokingham, UK) was used for primary antibody detection without a blocking step as per manufacturing guidelines. DAB was added and the sections were dehydrated in grades of ethanol 70%, 95% and 100% twice. Then Histoclear was applied before sections were mounted in DPX®. The degree of immunostaining was then quantified using densitometry.
Dear Priya. If I understand your question you just want to measure staining intensity as a measure for COX (COX4?) content? This is simple to do in ImageJ/FIJI.I guess you are staining with fluorochrome conjugated antibodies?
Quick guide:
The easiest way is just to use ROI and measure.
The more automated way is to fist define the ROI by using the WEKA plugin on a picture of the cellmembrane (e.g. stained against dystrophin). When that is done, you load your picture with the COX staining. Be sure that the ROI is set from the ROI set by WEKA and redirect the analysis to the COX-picture. Remember to save the ROI!!!
If you want to relate the staining intenstiy to fibertype you can do it in two ways:
Counterstain your sections and take a picture of (almost) the same place. Now add the ROI from the COX analyses to this picture. Set your treshold and read of the intensity to find your fibertypes. Be aware that some of the cox staining migth be visible. My advise is to use another fluorochrome than what you have used previously.
The easier way is to stain a neigthbour section and manually check which fibers are e.g. type 1. If you then draw a white dot in the fibers stained for your fibertype you can easily read of the Min/max value when you again measure staining intensity... (should be 255).
Hope you get it to work.
Kristoffer
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