Questions about immunofluorescence - (Jun/20/2007 )

A couple of general questions:
I am starting immunofluorescence for 2 antibodies at one. Once I mix the primary antibodies, can I reuse them? How many times and how long can I keep them at -20C?
I assume I cannot reuse secondary antibodies, is that correct?
Once I mount the cells, how long can I keep the slide before I go to the confocal microscope and under which conditions, e.g. temperature and do the slides need to be covered so that light doesn't penetrate.
Thanks for your help

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Ya, u can use primary ab again but keep it at 4C. Ya U cannot use secondary ab. If ur using secondary ab tagged with FITC, TRITC, Texas red, u can use antiquenching agent like PPD or Mowiovl (check the spelling). Slides can be kept at 4C.

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you can reuse primary and secondary Avb; primary often works better after the second or third re-use; secondary Ab should only be used once but can be re-used; the limiting factor is the label which may be less stable than the Ab itself;


store at 4°C plus 0.001% NaN3 for preservation

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Thanks for the help. What about storing the slides? How long can I store them and should they be covered?

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keep em in cardboard trays covered in aluminium foil at 4C they'll be good a week later, after two weeks you'll get blurring of the image
(assuming you keep them covered every second they are not under the microscope)

dom

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fix with mowiol as suggested, add DABCO to reduce fading of fluorescence; store at 4°C darken

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One can reuse primary and secondary antibodies. Store them in -20C and use even after months.

Keep the slides in 4C in a slide box and if you mounted it using media which prevent fluorescence bleaching, you can store it and use the slides for a few weeks without any major decrease in signal intensity.

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