Two very genral question regarding muscle fibers staining

AR · AR

Greetings to all,
ppl must have seen other topics from me regarding this matter. All of my other queries/problems are interrelated, but i am posing these questions separately to avoid any confusion.

Following are the questions:

1) While sectioning a rat soleus muscle on a particular day, i was having wrinkling in the sections (specimen temp = -21 C), so i had to decrease the temperature variably. In this process, i decreased it to first -24 (waited for ~10min, but still wrinkling. Then i decreased to -25, waited, sectioned; -27;waited, sectioned). At -27, wrinkling was absent but the sections started having cracks (due to low specimen temperature). Ultimately, i could obtain good sections at -22C (with no wrinkling and cracks). After staining (laminin), the section seemed grossly damaged. The fasicles and muscle fibers were distorted/displaced in shape throughout the section. Could it be the temperature changes that i did during sectioning? I have stained this very muscle before also with no such damage (ruling freezing artifact).

2) Can laminin background and/or freezing artifact kills the type2x staining (in rat's soleus muscle) using primary Ab = BF-35 (1/200) and secondary Ab = Alexa488 (1/200, green color)?

Hoping to hear from someone soon!
BEST,
AR

AR · AR

Q = Some time, while sectioning, cracks in the incoming sections occurs owing to low specimen temperature. I tried many combination of the chamber and specimen temperature, but, at times, the temperature of specimen just does not comes right...... I heard ppl saying that one can touch the specimen with his fingers for few seconds to increase the specimen temperature so as to bring down the specimen temperature to optimal.
my concern is does this cause temperature damage to the specimen???

Plzz reply!

Best,
AR

Carl · **Carl**

Hmmm, I had thought you would get some advice.
I admit that I do not know what is the answer for your cutting problem...

1. I can only suggest that the block of tissue has been damaged/defrosted and refrozen after the 1st succesful cutting? Such small temp changes in the cryostat should not cause such damage
2. No way, imho. Laminin is not co-localised. Any artefact will distort, not kill, any positivity.

In answer to your second post re touching...sure. Not for a few secs, though.
Just a quick wipe of the finger across the surface, then immediately cut.
Of course, Health and Safety must be considered before you consider doing this.
You can also, if the block is slightly warm, give it a short spray with "Polar" spray, to cool te block to enable several sections to be cut.
Cryostat sectioning can be the most difficult of all section cutting, imho.

Carl

AR · AR

Thanks Carl,
I thought i would not get any answers, considering the fact that i have quite a bit of questions in different posts here Confused

....... I have not proceed in my experiments as i wanted to know these answers.....

Only 3 problems are remaining:

1) This temperature damage problem (which is occasional and random. Since i am mounting the sections on slide in a homogenous way every time, i felt that, for some specimen, the change in cryostat and/or specimen temperature over a wide range might be the culprit.... you mentioned, it would not be causing problem.... ok...i should be ignoring this occasional problem as of now. )

2) Laminin background problem (Initially i was advised that laminin background kills the type2x staining. Considering your opinion and the fact the after omitting laminin and rhodaminin on the section, the type2x staining was still not positive. I have the images and will be posting here for your advice...)

Carl, now, i guess, the problem is very near of its solution.Do you mind if i write a summary of differential diagnosis of the type2x staining problem?......... I mean what could be the culprit and how they have been narrowed down to lesser number of culprits.

Thanks for taking the lead in here also!!........Hope other members will also share their opinions/experience
Thanks a ton
BEST,
AR Very Happy

Carl · **Carl**

NB: I stated that it is unlikely to be the cryostat if the temp fluctuation is over a narrow range, not a wide range, lol.
Imho, between -15 and -30 will not cause the block to deteriorate....it will just affect the quality of cutting, imho.

Your thoughts re x2 fibre staining problems would be interesting to read.
Best

carl

AR · AR

Hey Carl...!!

While thinking of the differential diagnosis of type2x problem, i happen to catch a very unusual thought. There is one more remote thought, but please provide your opinion on this one first.

1) I do PBS washing of the sections with syringe (filled with PBS) on one side of the section while simultaneously suctioning from the other side of the section.

2) I tend to touch the PBS (already over a section) with syringe tip and then do the usual suctioning (as mentioned in point 1). In other words, i use surface tension of the solution on the section rather than pouring PBS on the section in a drop by drop manner (which i feel creates agitation over the section and might result in damage).

3) I do washing of the type2x sections along - with all other sections (that contained both primary and/or secondary Abs for type 1, 2a, and 2b ). Is it a possibility that because i am touching the sections, the PBS in the syringe gets contaminated from type1,2a,2b antibodies. As a result, when i pour the same PBS on the type2x sections then the type2x Ab is blocked or competed by the contaminated Abs of type1, 2a, 2b.
In other words, the section for type2x staining not only has 1 and 2 Abs for type 2x, but also contains Abs of .....type1,2a,2b. This might result in some cross-reactions among the Abs....and ....no type 2x staining.

Please let me know what you think.
Eagerly waiting!

BEST
AR

Carl · **Carl**

I don't think so....the volume of the PBS is so much greater that the Abs ( contaminating ones) will be too diluted to have any effect.
You may be right however....I cannot be there in your Lab when you wash your sections, lol.

I think that your washing method is too complicated/time -consuming.
If you are worried that you will damage the sections, I reckon that there is more chance if you use a syringe so close to the section.
If you have correctly adhered your sections to the slide ( by airdrying under a fan after mounting onto a slide) the sections should not come loose/get damaged in washing.
I use a plastic wash bottle with wash buffer: hold the slide vertically with fingers on label end of slide and gently squeeze to get a stream of buffer washing down the slide. Then I add all slides to a rack in a large excess of buffer in a 500ml beaker. The rack is fixed suspended off the bottom with a 1ml pipette tip to allow for a magnetic stirrer bar underneath. Then I gently allow to stir on a magnetic stirrer, while I make up secondary Ab.
Sure, if you have multiple sections on your slides with different Abs/dilutions of one Ab, wash sideways!

Again, if you are concerned that some sort of interference is blocking type2x fibre staining, have you yet done a single Type 2x Ab incubation ( no other Abs), for those fibres??
Curious Carl

AR · AR

Again, if you are concerned that some sort of interference is blocking type2x fiber staining, have you yet done a single Type 2x Ab incubation ( no other Abs), for those fibers??

Reply : I have done only type2x previously, and also did just now, staining is still not positive....
Thanks Carl, at least things are narrowing down! I will be posting the list of type 2x staining problem argument/counterargument soon.

BEST
AR

Carl · **Carl**

Ta for quick answer.
Perhaps the Ab for 2x is no good for frozen sections?
Perhaps there are no 2x fibres in the muscle?

However, I recall stating that your Ab is also positive for other MHCs??

Sure, I could look this up but........I am sorting out teaching stuff and...listening again to some wonderful music by Agnes Obel ( her Philharmonics album... Smile

)))
After Agnes I will listen to the Most Excellent Emily Portman's album: The Glamoury!!!

Carless.... Wink

AR · AR

Hey Carl,

Perhaps the Ab for 2x is no good for frozen sections?
REPLY : People in my lab have done this type2x staining on frozen section before. However, the only difference between me and them is the type of secondary Ab used. Others used FITC and i am using ALEXA488. Since ALEXA 488 seems to be fine (as it is also used to stain type2a in my protocol and they stain beautifully), i do not doubt secondary Ab as a problem; however i am going to use FITC in my upcoming staining for type2x. In this way i will have everything same with my previous protocol that revealed type2x staining.

Perhaps there are no 2x fibres in the muscle?
REPLY: That is what i thought when i was using control muscles; however i have tried using samples which are sure to have a decent amount of type2x antigen/fiber. But i got almost the same kind of staining......... but this type2x staining is all but type2x, so i think that this staining should inevitably stains fibers (unless all the fibers are type 2x, which is rare)

I will soon display the possible problems and their feasibility/non-feasibility......

Let me know what you think about this.....until i post the D/D for the type2x problem...

Thanks a lot for being taking so much pain for my problem.....
GOD bless you!!

BEST,
AR

Carl · **Carl**

FITC will be as good as ALexa 488 for most apps . However, it is not as stable and bright as Alexa 488 so you are unlikely to get a better result.
However, who knows...we are often so constrained by our certainties that we do not try some seemingly illogical steps that sometimes give great results: who would have thought to stick a Pwax section in boiling solution?

Back to reality, lol: did you miss my statement about your BF-35 clone?
DSHB give this :

"Data Sheet: Click here
Antigen: myosin heavy chain, all but 2X
Contributor: Schiaffino, S.
Cells Available: Yes
Host Species: mouse
Isotype: IgG1
Antigen Species: bovine
Species Tested: rat, mouse, and human
Immunoblotting: yes
References: J. Muscle Res. Cell. Motil. 10(3), 197-205.; Asian-Aust. J. Anim. Sci. 24(1), 125-129. "

They clearly state that it does NOT demonstrate type 2x MHC.

Waddya fink?

Best
Carl

AR · AR

Reply with quote
FITC will be as good as ALexa 488 for most apps . However, it is not as stable and bright as Alexa 488 so you are unlikely to get a better result.
However, who knows...we are often so constrained by our certainties that we do not try some seemingly illogical steps that sometimes give great results: who would have thought to stick a Pwax section in boiling solution?

Back to reality, lol: did you miss my statement about your BF-35 clone?
DSHB give this :

"Data Sheet: Click here
Antigen: myosin heavy chain, all but 2X
Contributor: Schiaffino, S.
Cells Available: Yes
Host Species: mouse
Isotype: IgG1
Antigen Species: bovine
Species Tested: rat, mouse, and human
Immunoblotting: yes
References: J. Muscle Res. Cell. Motil. 10(3), 197-205.; Asian-Aust. J. Anim. Sci. 24(1), 125-129. "

They clearly state that it does NOT demonstrate type 2x MHC.

Waddya fink?

REPLY : Thanks Carl for your prompt reply!
I completely remember you telling me the link where we can get information about the Abs. The details of Ab you mentioned above and the link (which i cannot open) is, i am pretty sure, the same from where i bought the BF-35 Abs.

I am not sure what you have asked me here, but i assume that you allude to the fact that BF-35 do not stain type2x, so i would not be getting any type2x positive staining.
I agree with you; however i should have , at least, other fibers staining positive (green in color) even if there is not type2x fibers in the section.

Please reply!
BEST
AR

Carl · **Carl**

You are correct, one would expect all MHC, excepting 2x to be positive.
Thus, any negative fibres would be 2x?
However, the datasheet only states that it works on immunoblot .
It would be interesting to see an image of your colleague that got this Ab to work on frozen sections!
I assume that it would be an image showing most fibres positive with occassional fibres negative ( Type 2x).
Can you post one?
Best...

Carl

AR · AR

Hey Carl,

1)I happen to get the address/site from which i ordered the BF-35. Please find the data sheet on this link (it is right there in the center of the screen). The data sheet mentions that it is used for immunohistochemistry.

http://dshb.biology.uiowa.edu/myosin-heavy-chain-all-but-2X

2) Attached is the image from one of my lab members. Type2x image in the control soleus (the same type of muscle i am using)

http://www.immunoportal.com/modules.php?name=gallery2&g2_itemId=18210

Waiting for your appraisal
BEST
AR

Carl · **Carl**

Your lab member image of muscle is very good!
Sure, it's enhanced.
However, I see no negatively stained fibres in the BF-35 image....thus no Type 2x fibres?

Also, I draw your attention t o this comment of yours, in an early post:

" Can laminin background and/or freezing artifact kills the type2x staining (in rat's soleus muscle) using primary Ab = BF-35 (1/200) and secondary Ab = Alexa488 (1/200, green color)? "

It implies that something is killing type 2x staining......however, BF-35 will never stain Type 2x fibres.

Curious Carl

Immunohistochemistry	In Situ Hybridization	Immunostaining
Immunohistochemistry Images	Tissue staining	Pathology
Tumor Markers	IHC Reagents	Tissue Micro Array
Immunofluorescence Staining	Positive Control Slide	Polyclonal Antibody
Monoclonal Antibody	IHC Staining Protocol	In Situ Hybridization Images
Immunohistochemistry Protocol	Immunofluorescence	Immunohistochemical Staining
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