[Histonet] MIXED fiber type identification by Immunohistochemistry

[arjun ruhella]

Greetings to all, 

In immunohistochemistry, I am encountering confusion in designating muscle fibers (based on staining color intensity) to a particular fiber type. 

This problem is particularly bothersome when i encounter the fibers stained with 2 different colors, one color has good intensity while other seems to be of light intensity. I don't have any criterion whether to consider this light staining as actual staining, which would classify the fibers as MIXED fibers or should i consider this light staining as background/non specific staining in which case the fibers will be classified as PURE fibers.   

Does anybody can suggest any criterion/tips they have been using to identify muscle fiber type as correctly as possible? I can understand that identifying MIXED fibers is subjective in immunohistochemistry, but it would be of immense help if i could get to know of any objective suggestions.


Animal= spinal cord injured Rat
Muscle = Soleus
Section Thickness = 10µm 

I will really appreciate the help! 

Thanks, hope to hear from ppl soon! 

BEST, 

AR