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REPLY: 1) Sure Carl.....i guess this type2x staining nomenclature is causing some confusion......i completely understand that this is all except type2x fibers.
2) Since the above mentioned image is a control/normal soleus muscle, type 2x is less likely to present; however, i am not even getting the other types of fibers stained in the section.
BEST
AR
Sun Sep 18, 2011 7:18 pm
AR
Rank: Member
Joined: Jun 10, 2011
Posts: 59
Post subject: type2x staining problem solution - differential diagnosis
Hi Carl,
I have written the type2x problem keeping in mind the crucial steps in my protocol i.e. antigen, fixation, blocking, primary Ab, secondary Ab. The steps are written in number like 1, 2 .... and under each steps i have the hypothesis of the problem and argument for and against the hypothesis.
For
• Acetone and methanol have been purported to mask antigenicity. May be this type2x antigen is very sensitive to the fixatives I use.
Against
• MHC proteins are pretty robust and do not get damage easily
• If fixation can cause antigen masking/damages, then it is unlikely to get positive staining for other fiber types.
• People have obtained good type2x staining with the same fixative that I am using.
3. Blocking
Hypothesis: Cannot think of any problem associated with this
except blocking time is less
Against
• Has increased blocking time from 1hr to 2hr, but no positive staining
4. Primary antibody or 10 Ab (mouse anti-MHC type 1, 2a, 2b IgG1)
- Hypothesis: 10 Ab is inactive/degenerated
Against
• Have already ordered 2 times the new BF-35 Ab
For
• Abs are cheap and, at times, are not of good quality
- Hypothesis: 10 Ab is non-specific
For:
Can’t say: Tried 3 combinations
1st section = no10 Ab + no 20 Ab = section was more or less dark under fluroscent microscope
2nd section = no 10 Ab + 20 Ab = section was more or less dark under fluroscent microscope
3rd section = added nothing, just looked the section under fluroscent microscope immediately after fixation. Section was more or less dark under fluroscent microscope
- Hypothesis: 10 Ab is big in size so cannot diffuse inside the fibers i.e. permeability issues.
Against
• Type2a 10 Ab is also IgG1 like BF-35, and I get good staining of type2a fibers (Fig 2) Therefore IgG1 Ab can diffuse in to the fibers
5. Secondary antibody / 20 Ab (Alexa 488 goat anti mouse MHC IgG)
Hypothesis: 20 Ab is inactive/degenerated/non-specific
Against:
• Alexa488 is use to stain type2a also, so Ab is active. Moreover, when looked in green filter, the green color is very specifically localized to the stained fibers with rest of the area looking dark/muddy color thus, ruling out non-specificity (Fig 2 = http://www.immunoportal.com/modules.php?name=gallery2&g2_itemId=18217).
6. 10 Ab and 20 Ab incompatibility
Hypothesis: 20 Ab is IgG whereas 10 Ab is IgG1 isotype. Since secondary is not specific to primary isotype, staining is not coming positive.
- Freezing artifacts
Against
• no positive staining with muscles without freezing artifacts
- Laminin bleeding/background
Against
• No staining when the laminin and/or rhodamine concentration is decreased
• No staining when laminin and rhodamine is omitted i.e. only type2x staining without laminin staining (Fig 1).
- Autoflorescence/non-specific binding
• With simultaneous autoflorescence or non-specific binding, there should be some positively stained fibers (obviously with background).
Possible solution:
1) Use of FITC
2) Order new BF-35 again
3) Use of triton X 100 for increasing permeability of fibers
Thu Sep 22, 2011 3:35 pm
AR
Rank: Member
Joined: Jun 10, 2011
Posts: 59
Post subject:
Hey Carl,
I know my last post (differential diagnosis of type2x staining) was pretty lengthy, but i was just wondering if you get a chance to look at it. I really need your advice on this aspect.
Waiting for your reply!
Thanks
AR
Fri Sep 30, 2011 5:00 pm
Carl
Rank: Member
Joined: Oct 11, 2004
Posts: 1871
Post subject:
Hey AR...
my apologies.
I missed your posts...preparing for new year student teaching so, got distracted.
Ok, I will read carefully through your seemingly comprehensive Dialectic
But, while I am doing this..
1. Please, is there anyone out there who can also help?
I know some stuff but I will admit to being an amateur....not an expert.
Ian, where are you in my time of great Need????
2. AR.....am I correct in stating that you get all Abs working very well except BF-35??
K..back to reading my Histology Workshop notes for Tuesday, sigh.
Can do it on the weekend but....as we in Engerland are having some weird stuff called Sunshine, I reckon I'd better get some rays down at the beach tomorrow
Have a damn fine weekend, AR.
Respect,
Carl.
Fri Sep 30, 2011 7:21 pm
AR
Rank: Member
Joined: Jun 10, 2011
Posts: 59
Post subject:
Carl!
Thanks for the reply......
Please take your time, have a wonderful weekend!
P.S: you are right, my all Abs work fine except tye2x
I was to lazy to read all the stuff. Am I correct, that this BF-35 antibody should label your type 2 fibers?
I've read this sentence in the article above: "These fibers were also labeled by BF-35 antibody, which recognizes all MyHC isoforms, except -2x."
There are so many fibertypes and antibodies mentioned....
Perhaps your staining problem is more a kind of test-setup problem.
Many thanks for your very interesting comments and links.
Respect, you Lazy git!
Batch variation is often a problem......
However, AR....I asked if BF-35 stains fibres.
It should stain fibres...?
If it does not stain fibres then....Gula is correct?
ie: all of your samples of BF-35 are useless?
If all of your other Abs are working very well, it is not your technique, as Gula indicated?
Yes...a damn fine Saturday.
For Engerland, we had exceptionally good Sun at this time of year.
I tasted Strawberry-flavoured cider for the first time, at our local Food festival...it went very well with a Venison Burger with Bacon, Rocket and Haloumi cheese
The Strawberry cider reminded me of Kriekbeer, if I recall it correctly?
Carl
Sat Oct 01, 2011 6:22 pm
AR
Rank: Member
Joined: Jun 10, 2011
Posts: 59
Post subject:
Thanks Gula and Carl for coming up with the advice!
I really appreciate your help.
I will see in to this articles.
Carl - my BF-35 Ab should stain fibers
BEST,
AR
Mon Oct 03, 2011 2:00 pm
AR
Rank: Member
Joined: Jun 10, 2011
Posts: 59
Post subject: Got POSITIVE type2x staining :-)
Hey Carl, Gula
I got type2x positive staining positive!
I used the same stock of Abs, but played with the concentration of primary a lot. At the end, the positive staining came at a dilution of 1/5 as opposed to 1/200 in my protocol.
I cannot think of going down to this much concentrated primary Ab.
As of now, i don't know, but the option of uploading the image is not coming on this site. May be will try later on and post one of the sample image.
I am sooo thankful to your advices. I really learned a lot!
Thanks a ton!
Though muscle has lot of freezing artifact, but ,i also had some more positive staining along with this.
BEST,
AR
Wed Oct 26, 2011 11:34 am
Carl
Rank: Member
Joined: Oct 11, 2004
Posts: 1871
Post subject: Congratulations, AR.
The signal: noise is not good but, the fibres are still obvious!
I suppose that, as it is a TCS, the titre of Ab is not high.
TCS must sometimes be applied neat.
Or, the Ab has a low affinity.
Of course, using a stAvidin conjugated fluorochrome ( after biotinylated secondary) you should be able to increase signal: noise/obtain a better dilution factor.
Sure, using Tyramide amplification will increase this ~ 2-10 fold.
NB: Buying from DSHB, BF-35 will cost only ~$32 for 1ml!!
Best
Carl
Wed Oct 26, 2011 5:28 pm
AR
Rank: Member
Joined: Jun 10, 2011
Posts: 59
Post subject:
My apologies Carl, but i did not understand the abbreviation you mentioned i.e. TCS . Still naive with immuno!
BEST
AR
Thu Oct 27, 2011 5:15 am
Carl
Rank: Member
Joined: Oct 11, 2004
Posts: 1871
Post subject: Soreee, AR
TCS= Tissue Culture Supernatant.
Monoclonal Abs are mostly generated by cells in culture ( the cells secrete the Ab into the TCS).
Sure, to get a concentrated Ab, the cells are injected into animal peritoneum...the cells grow ( a tumour) and express fluid -ascites- into the peritoneal space.
Not nice for the animal but...what is "nice " for exptl animals???
However, the Ab titre is much more concentrated.
Sure, one can concentrate Abs in different ways .....using Ammonium sulphate ( all Abs).
A less crude concentration will be obtained by using Protein A/G to pull all IgGs out.
However, the "best" way is to run the fluid down a column which has the antigen covalently bonded to it: only Abs against that particular Ag will stick to the column.
Then, the Ab is eluted ( released) from the column.
You end up with a buffer containing Ab that is ONLY directed against that Ag ( an immunogen affinity purified Ab)
However, it can be argued that the Abs with the highest affinity for the Ag ( the "best") are left on the column....
So, if you use "normal" serum as blocking control/or added to primary Ab it is a waste of time if Primary/secondary Abs are purified in this way.....because the Ab diluent is not serum..it is buffer
Imho, pre-immune serum from the same animal, if the Ab you buy is serum, is the only valid negative control.
So many variables!
Confused Carl
Sure, I get details wrong so, I am happy for anyone to correct my poor explanations.
BTW: you are doing well, AR.
You are willing to share your ignorance.
It is a damn fine quality.
Carl
Thu Oct 27, 2011 5:18 pm
AR
Rank: Member
Joined: Jun 10, 2011
Posts: 59
Post subject:
Carl,
Thanks for the explanation!
I really appreciate yours and others in this forum for helping novice ppl like me.
Hope to be contributory to this site and also gain more knowledge from here.
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