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Problems with Immunostaining


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#1 Debbie_M

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Posted 21 December 2010 - 02:00 PM

Hi guys!

I am posting because over the last three weeks I have failed to get any data from microscopy. My problem is that the BrdU incorporation/immunostaining is not working. I have been doing this protocol for the last 4 months without any problems using U2OS Cell line. Recently, every time I have been takeing the slides I made down to the microscope, the BrdU staining is all over the field of view and it looks the nucleus has "burst" and the DNA is spread all over the coverslip. I repeated it all again today using fresh reagents and this time I couldn't see anything at all. I should also note that I have failed to stain the cells with Dapi on all of these occasions and although I can see a slight blue signal when looking down the microscope, I cannot focus in on a cell!

I would really appreciate some feedback/suggestions! If you need anymore information let me know!

Thank you in advance for any help!

Debbie

#2 bob1

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Posted 22 December 2010 - 12:56 PM

How did you permeabilise the cells for DAPI/brdu detection?

How are your antibodies (especially the flourescent ones ) stored?

#3 Debbie_M

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Posted 23 December 2010 - 04:31 AM

How did you permeabilise the cells for DAPI/brdu detection?

I fix and permeabilise the cells using 70 % ethanol for 30 minutes at room temperature

How are your antibodies (especially the flourescent ones ) stored?


The BrdU primary antibody is stored at 4 degrees (Fridge)
The Alexafluor 488 secondary antibody is stored in a light sensitive tube at 4 degrees
DAPI is also stored in a light sensitive tube at 4 degrees

#4 bob1

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Posted 04 January 2011 - 12:59 PM

Your storage looks fine, though I would store the 488 aliquotted and frozen at -20. How did you permeabilise the cells?

#5 Debbie_M

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Posted 04 January 2011 - 02:17 PM

Your storage looks fine, though I would store the 488 aliquotted and frozen at -20. How did you permeabilise the cells?

Ethanol both fixes and permeabilises the cells.

#6 bob1

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Posted 05 January 2011 - 12:44 PM

Not really, try using 0.5% triton-X100 in TBS or PBS for 10 minutes to permeabilise.




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