Background in immunostaining. Help.

To: Nikolai troianovsk_s at MSDISK.WUSTL.EDU
Sat Aug 5 13:32:42 EST 1995


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Date: 5 Aug 1995 15:03:23 GMT
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Message-ID: <40017r$n67 at galactica.galactica.it>
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Dear fellow scientists,
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I'm trying to immunostain Myelin with the serum of a patients having
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serum's IgMs toward a myelin-specific glycoprotein.
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However, my second, FITC Ab always give me serious problem of background,
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even at a 1:3000 dilution. 
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Any suggestion would be greatly appreciated
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Hi, there.
Try the following prot.:

Before fixation steps, always wash cells out from serum soaking them (or by
other means) in PBS. Then proceed to fixation.

5 min MetOH at -20oC;
1 min Acetone at -20oC;
Let them dry at RT. (The cells could be stored at this stage for a while)

Dried cells before staining should be always wetted by PBS w/o any serum!!!
Then proceed to staining.
After the staining wash sells from secondary Ab in PBS, then wash from salt
rising cells with water (several sec.) and then put cells in EtOH (98%) for
1 min at RT. Let them dry out, they are ready.
I use this prot. to stain components of cellular membranes. Dono about myelin.

good luck.

Nikolai
http://128.252.119.253





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