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following are the details:
1) sample = rat's soleus muscle
2) primary Ab = BF-35 (mouse IgG1) (1:200)
3)secondary Ab = Alexa488 (goat anti - mouse IgG) (1:200)
I have been told that it is a negative staining as individual muscle fibers cannot be deciphered. Also, i am clueless of the cause of this kind of staining.
Please provide your guidance, i am stuck at bench!
Best,
AR
Thu Aug 18, 2011 5:51 pm
Carl
Rank: Member
Joined: Oct 11, 2004
Posts: 1871
Post subject: Hmmm...more problemos, AR ;-)
Have you immunostained using anti slow MHC?
I would like to know what result you get with that.
Carl
Fri Aug 19, 2011 3:57 am
AR
Rank: Member
Joined: Jun 10, 2011
Posts: 59
Post subject:
Hi Carl!
Thanks for being with this problem too. I am trying to solve these problems at the earliest, so posting two messages simultaneously.
What i understand from you question "Have you immunostained using anti slow MHC? " is - what results i get when i stained the same muscle section (or consecutive section) for type1 muscle fibers?
Ans: type1 fibers staining is decent, as type1 fibers are stained blue in color and blue color images/stain is always coming light (stain intensity) with my staining. Following are the Abs i used for type1:
Primary = BA-D5 (mouse IgG2b)
secondary = Alexa 350 (a21140) (goat anti-mouse IgG2b)
Color produced = blue
Eagerly waiting for your guidance!
Best,
AR
Sat Aug 20, 2011 4:57 pm
Carl
Rank: Member
Joined: Oct 11, 2004
Posts: 1871
Post subject: Ta for info, AR.
OK, if type 1 fibres are immunostained good, your method is fine.
BF-35 antibody: check out DSHB's website and note that they state that this Ab is for all MHC EXCEPT Type IIx!
http://dshb.biology.uiowa.edu/
An excellent resource : cheap Abs!!!
I always check them out before I look elsewhere for Ms monoclonals.
Remember that you are using mouse on rat and any endogenous IgG will x-react unless the secondary is rat IgG - adsorbed.
( I am very grateful to Hogne for informing me
Unless you are doing triple labelling and no nuclear staining, why do you want to use Alexa 350?
Also, unless you have a specific desire/requirement to use isotype-specific secondary Abs, it will be much cheaper to use anti IgG ( so, if your primary is IgG2a and you are only using one mouse monoclonal, do not bother to use anti IgG2a......same applies for any other species of Ab;-)
I hope I am right and clear in my answer so, anyone else, please correct me if I am wrong.
Carl[/i]
Sun Aug 21, 2011 4:40 pm
AR
Rank: Member
Joined: Jun 10, 2011
Posts: 59
Post subject:
OK, if type 1 fibres are immunostained good, your method is fine.
BF-35 antibody: check out DSHB's website and note that they state that this Ab is for all MHC EXCEPT Type IIx!
Reply: you are right Carl, sorry about writing type2x staining always. It is all except type2x staining.
http://dshb.biology.uiowa.edu/
An excellent resource : cheap Abs!!!
I always check them out before I look elsewhere for Ms monoclonals.
Remember that you are using mouse on rat and any endogenous IgG will x-react unless the secondary is rat IgG - adsorbed.
( I am very grateful to Hogne for informing me Smile
Reply: Endogenous X-reaction is what i initially thought. I know a little about the concept of adsorbed secondary Abs, and this has not been attemped in our lab before. Ppl have just stained type2x without carrying out adsorption of secondary Abs. So, my potential solutions for this type of staining are: increase the blocking time (from1 to ~2hrs), more washing for the section stained for this particular stain (but i am already doing extensive washing), decrease Abs concentration (dicy), change secondary Ab to specific epitope ,IgG2a (you mentioned, it might not be of much help)...rest i am clueless ...provide your experience plzz.
Unless you are doing triple labelling and no nuclear staining, why do you want to use Alexa 350?
Reply: Alexa 350 has been used in my lab before by others. Other option is FITC, which i might try if i cannot figure out problem with Alexa350. The reason i want to use Alexa is that it produce green color and i can then use this green color Ab with other blue color secondary Ab (type1) to stain two fiber types on a single section thus, reducing some work. On the other hand FITC gives green color so in order to stain each of four fiber types i would need to stain four sections anyhow.
Also, unless you have a specific desire/requirement to use isotype-specific secondary Abs, it will be much cheaper to use anti IgG ( so, if your primary is IgG2a and you are only using one mouse monoclonal, do not bother to use anti IgG2a......same applies for any other species of Ab;-)
I hope I am right and clear in my answer so, anyone else, please correct me if I am wrong.
Carl[/i]
Sun Aug 21, 2011 11:22 pm
Carl
Rank: Member
Joined: Oct 11, 2004
Posts: 1871
Post subject: Thanks for more info...
Primary Abs: If I am using a mouse Ab on rat tissue and the tissue is not perfuse-fixed I will use a rat IgG-adsorbed secondary Ab.
This is purchased, not prepared in my lab.
Check out Vectorlabs for adsorbed biotin conjugated secondaries, for DAB for eg, or Invitrogen for adsorbed Alexa dyes.
I mention biotin conjugates as one can use streptavidin-488/594, for example, for increased sensitivity ( thus you can further dilute your precious primary Abs
Re fluorochromes: as I said, if you can afford it, use Alexa 594 ( red), Alexa 488 ( green).....then if you do not care to IF-stain nuclei using DAPI or Hoechst also use Alexa 350 ( blue).
Sure, you can also use Cy5 or Alexa 650 if you have infra red filters.
Of course, FITC and TRITC are good...
Carl
Mon Aug 22, 2011 4:58 pm
AR
Rank: Member
Joined: Jun 10, 2011
Posts: 59
Post subject:
Carl,
Whatever you explained was clear to me;however, i might need to read more about adsorbed secondary Ab in order to discuss and solve this type2x problem better.
Will get back to this query later after reading.
Thanks
Best,
AR
Wed Aug 24, 2011 12:28 pm
AR
Rank: Member
Joined: Jun 10, 2011
Posts: 59
Post subject: A general question for clarification
Hi Carl,
Just wanted to ask:
if i am conducting a negative control staining (without pouring primary Ab , but pouring secondary Ab), and i see the section under florescent microscope, does the section should look completely dark (assuming there is no non-specific binding and auto-fluorescence)
Reason of asking = I did not pour primary Ab for type2x, but pour the secondary Ab (producing green color). The muscle fibers are more or less dark, but the borders of muscle fibers and the interstitial space show green color when looked in the microscope. Does it mean that the secondary Ab is non-specifically in binding to the fibers borders and to intrinsic IgG in interstitial spaces of the section?
Waiting for your reply!
BEST,
AR
Wed Aug 31, 2011 1:31 am
Carl
Rank: Member
Joined: Oct 11, 2004
Posts: 1871
Post subject: Hi AR.
You are asking good Qs!
If you include a "no primary" control but, applying ( not pouring;-) secondary Ab, you should also include a "no primary/no secondary" control.
You will then be sure that any fluorescence seen is because of primary, secondary OR because of endogenous fluorescence!
You will then compare 1st slide of primary/secondary...second slide of no primary with secondary....and third slide of no primary /no secondary.
The 1st slide should give you strong specific fluorescence, compared with a weaker endogenous /background fluorescence EXCEPT where endogenous IgGs are present, which will be as strong as your primary Ab immunofluorescence.
I apologise if I am not clear as I can only communicate in English.
If there are other members who can make what I say more clear, or even show that I am making mistakes, I would be most grateful.
Carl
Wed Aug 31, 2011 4:14 pm
AR
Rank: Member
Joined: Jun 10, 2011
Posts: 59
Post subject:
Carl ! asking good questions ...
Your explanation is, as usual, self-explanatory!
Thanks for being so active n this forum to help others like me.
I wish i would be able to contribute to this forum one day.,.....
Best,
AR
Thu Sep 01, 2011 1:21 pm
Carl
Rank: Member
Joined: Oct 11, 2004
Posts: 1871
Post subject: Thanks, AR
You ARE contributing by asking important Qs!
It will help other Newbies and....it focuses the mind of us so-called "experts" to address basic Qs in a simple way.
Well, I can only speak for myself and you have managed to focus me )
I am also learning from others.
I can only attempt to see further by standing on the shoulders of experts who not only came before me but, also those who are still alive and kicking!
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