[Cell-biology] Re: Mitotic spindle fiber stain
phoenixhou at gmail.com
Mon Apr 10 03:37:34 EST 2006
Uh...Well, maybe there are commonly 3 ways to stain microtubules as
well as other components of cytoskeleton like F-actins. It is the
purpose of you experiment that decides.
1.Stains with labelled probe,usually fluorescence labelled.It is
readily obtain from many vendors like Invitrogen or Sigma.In most
occasions,you can get a very nice photo from that.However it will cost
you much,and the standard protocols are not directly applicable-I
suffered from this recently so believe me.And the main drawback is you
can hardly stain a cell while it's living.So this method is eligable in
morpholgical research but unavailable when you want to do vital stain.
2.Immunofluorescence.Average cost,average effort required and quite
nice photos obtainable(by quite nice I mean it surely do some harm to
cells).There are tons of protocols about this method.I feel strange why
you aviod it.But it is you make decision.
3.GFP,recently GFP-mTn.As to label mitotic spindles,which mainly
contain microtubules,you could construct a fusion protein with
alpha-tubulin and GFP/GFP-mTn,express it in vivo,excite it,say cheese!
It's a perfect method because least harm,if not none,is done to
cells.Not only that,also there's no need trying to persuade your boss
to pay the bill for the kit,and you don't have to kill lovely bunnies
only to prepare antibodies.There are many articles on PubMed to which
you could refer.
Hope it would be useful.
I am now involved in a work on actin cytoskeletonin plant.Unfortuately
I am experiencing protocol frustrations.Fluorescence phalloidin from
Invitrogen sucks.The protocol enclosed is somewhat a piece of bullshit.
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