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In situ hybridization - huge background on slide where the paraffin was?!

in situ hybridization

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#1 Maya1988

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Posted 23 November 2012 - 02:48 AM

Dear all,

Once again it's me - I am having huge problems with my ISH. I cut the 4%PFA fixed tissues (embedded by myself in 100% clean, fresh, melted paraffin) onto poly lysine slides, dry them at 37 oC overnight and then go through extensive deparaffinization steps with xylenes (all fresh) and ethanols...however, I am getting humongous background!!! All around the sections, exactly in the shape of the original paraffin slice...

I observe this in nearly all my slides however not in ALL of them! How can one slide be completely clean, but so many others are dirty? And no, none of them dried overnight, all of them were treated in the same batch!

Did anyone ever face this problem? What to do? I am really at the end of my wit here and since this is so random and does not occur in all my slides, I don't even know how to best trouble shoot...

T.T

Maya

#2 bob1

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Posted 23 November 2012 - 11:46 PM

Try washing several times in 0.1 M glycine in PBS for at least 30 min each, preferably overnight. This will soak up free aldehydes left behind by the fixation process.

#3 Maya1988

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Posted 27 November 2012 - 06:15 AM

Dear bob1,

Thank you for getting back to me. At which step do you suggest doing this wash? I am doing fixation overnight when I collect the tissue, and then there are two more fixation steps in my protocol (already after the deparaffinization steps).

Best,
Maya

#4 bob1

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Posted 27 November 2012 - 12:17 PM

Why are you fixing more? This shouldn't be necessary and may interfere with the detection of some signals.

I would do these washes after any formaldehyde based fixation steps, but you could keep it until after the last fix.

As it seems that some of the signal is due to the paraffin, perhaps you need to change the brand as some have impurities that could be giving you the backgrround.

Do you see the same background in the actual tissue?





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