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Post subject: advice on identifying muscle fiber types on stained image
Greetings to all,
I am encountering confusion in designating a fiber (based on staining color intensity) to a particular fiber type. I have marked the controversial fibers with ARROWS in my images. Since I am staining serial sections, the arrow-marked fibers in FIRST and SECOND section are exactly same.
Question
In FIRST section = some ARROW-marked fibers in blue colored filter are stained lightly relative to rest of fibers. Should i consider them positive ?
In SECOND section = These same fibers are also stained green, but the staining intensity is relatively less when compare to other green stained fibers, however decent green color is present.
- Should i consider these fibers as mixed or either type ?
- Is their any criterion that someone can suggest to avoid this problem while doing fiber type % analyses. This way i can avoid any mistake in obtaining fiber type distribution in a muscle.
Type1 - Alex 350 (A21140) (Goat anti-mouse MHC1 )
Type2a - Alex 488 (Goat anti-mouse MHC2a)
Type2b - Alex 350 (A31552)(Goat anti-mouse MHC2b)
Fri Nov 11, 2011 1:18 pm
Carl
Rank: Member
Joined: Oct 11, 2004
Posts: 1871
Post subject: SC-71...
.....where from??
Not Santa Cruz.
Where from?
Thanks for info, AR.
I want to read the datasheets before I make any comments...
except to state that there may be subtle/major changes in MHC phenotype in muscles, that is dependant upon stress factors.
Are your specimens wild-type muscles?
Maybe there is up/down regulation of particular isoforms??
I have to check this.
So....has anyone else, PLEASE, got any answers/ideas re AR's post?
I am not sure, I have to admit.
Fri Nov 11, 2011 9:25 pm
AR
Rank: Member
Joined: Jun 10, 2011
Posts: 59
Post subject:
Carl,
All primary Abs, including SC-71, are from "Developmental Studies Hybridoma Bank". Here is its link:
Just to mention, Literature says that fiber type % distribution in control SOL of my rats species are variable. It can be-
90% type1, 10% type2a
Some report ~15% mixed fibers (1+2a)
I am concerned that , just because of being doubtful in interpretation- due to staining intensities, i may include pure fibers as mixed or vice versa.
Hope to hear your's and other experts advice on this issue!
Thanks
BEST,
AR
Sat Nov 12, 2011 3:58 am
Carl
Rank: Member
Joined: Oct 11, 2004
Posts: 1871
Post subject: Apologies for delay, AR
I am not "expert" enough so, I have asked the Muscle expert at Guys Campus for her expert opinion.
I shall post it here, when it is complete.
Soon
Carl
Wed Nov 16, 2011 6:32 pm
Carl
Rank: Member
Joined: Oct 11, 2004
Posts: 1871
Post subject: An expert's opinion.
From Dr Rowlerson, Senior Lecturer, King Colleg London:
First: rats have all of the ‘pure’ fibre types I, IIA, IIX and IIB. So in a muscle with lots of fast fibres there ought to be a few fibres which are negative with all the Ab (the IIX).
In addition, it is not unusual to have hybrid fibres co-expressing 2 (or more) myosins;
IIC (type I and a fast, usually IIA)
IIAX (IIA and IIX)
IIXB (IIX and IIB)
So probably the intermediate staining fibres are hybrids ....
The arrowed ones are presumably IIC
As far as I can see the ringed ones are similar (i.e. IIC)
From the fibre type composition and the shape of the fibres, I am guessing that the muscle in question is soleus ? This is 70-80 % type I, with some IIC and IIA (exact proportions depend on age of the animal).
The difference in %s is just because of the age issue I mentioned. There is conversion of many fibres from type IIA to type I (via IIC) over a period from about 1m to 3m postnatally. The older the rat, the more type I in soleus (though it probably won’t ever reach 100%).
Otherwise, I can’t see the problem – mostly the Ab are binding to the cut surface of the fibres anyway. There is no sensitivity problem with myosins – they account for about 30% of the dry weight of the fibre, so the amount present is vast. Intermediate staining means intermediate amounts (although I don’t think it is a linear relation – needs just a minor proportion of a myosin for a fibre to light up).
Thu Nov 17, 2011 4:55 am
AR
Rank: Member
Joined: Jun 10, 2011
Posts: 59
Post subject:
Thanks Carl for the answer!
Yes, the muscle in question is soleus of rat (age = 16 weeks).
I really appreciate you effort!
Thanks once again.
BEST,
AR
Sun Nov 20, 2011 10:38 pm
Carl
Rank: Member
Joined: Oct 11, 2004
Posts: 1871
Post subject: Are you satisfied?
AR?
Did our KCL expert in muscle ( and many other areas) give you ....satisfaction?
Kris Karl.
Fri Dec 23, 2011 7:03 pm
AR
Rank: Member
Joined: Jun 10, 2011
Posts: 59
Post subject:
Hey Carl!
My apologies, but i do not understand - "our KCL expert in muscle"
Respectfully,
AR
Tue Dec 27, 2011 8:24 pm
Carl
Rank: Member
Joined: Oct 11, 2004
Posts: 1871
Post subject: My apologies!
AR: I was just wondering if the info I obtained from Dr Rowlerson was actually useful to you?
Best wishes,
Carl
Wed Dec 28, 2011 4:48 pm
AR
Rank: Member
Joined: Jun 10, 2011
Posts: 59
Post subject:
Hi Carl,
Yes indeed! The information you provided me with was helpful.
Thanks a lot for that!
Respectfully,
Arjun
Hope all the health and happiness in this new year for you !
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