isolating nuclei - how to address sticky nuclei (Jan/26/2006 )

i have been isolating whole nuclei to subsequently fix and stain. but they are so sticky that i cannot get any single nuclei imaged. the end result must be in xylene but the issue is way before that.
i am looking at adding DNAse1, and /or cytochalasin B.
i am running out of ideas and deadlines are approaching.
any help or ideas.
the isolation is good, i am getting what i want. but i am concerned with cytoplasmic fragments left and am wondering if this is making it stick or i am sure released dna. so if that is the case, how to get rid of it.
mrcrawford11@yahoo.com

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how are you isolating nuclei? hypotonic treatment and 3:1 methanol acetic acid fixation?

if you are doing that you can reduce your KCl concentration, increase the time of hypotonic treatment, and reduce the amount of prefix you use. reducing the prefix helps to get rid of cytoplasm. also make sure your nuclei are well suspended before you fix, your cells are diluted enough, and you are not dropping on the same spot on the slide more than once.

washing with .1% triton x -100 and proteinase k treatment for a short time could help once the slides are dropped.

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i am using a hypotonic solution and the fixation is 2% PFA. the cells are left in suspension and are not put on a slide. not sure what you mean by prefix. the nuclei look good before the fixation. i go through an alcohol hydration step as all the water must be removed before the last xylene step.
i have not used KCL though i am aware of it. what is your concentration recommendation and please explain the prefix. thanks

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to isolate nuclei/chromosomes we use the following:

collect cells in a 15ml polypropylene centrifuge tube
wash w/ pbs + spin down
aspirate as much pbs as possible, then completely resuspend cells in the remaining pbs by gently flicking the bottom of the tube
once cells are resuspended add 7-10 ml of .4%KCl in dd water, invert tube to make sure the cells are distributed throughout and incubate for 20 min in a 37 degree water bath
after incubation add 100ul of 3:1 methanol:acetic acid to your tube and invert a couple times (prefix)
spin down at 800 X g for 10 min
remove as much solution as possible without disturbing nuclei
gently resuspend nuclei by tapping the bottom of the tube
add 7 ml 3:1 methanol acetic acid for 10 min @ room temp
spin down at 800 X g for 10 min
remove as much solution as possible without disturbing nuclei
gently resuspend nuclei by tapping the bottom of the tube
add 7 ml 3:1 methanol acetic acid for 10 min @ room temp
spin down at 800 X g for 10 min
remove as much solution as possible without disturbing nuclei
gently resuspend nuclei by tapping the bottom of the tube
add 3:1 methanol acetic acid until solution is just barely milky looking and make sure cells are well suspended
then drop 20ul onto a ice cold microscope slide
to drop hold your pipettor 6-12 inches above the slide and squeeze the solution out. you should get 2 drops with 20ul, make sure they don't land on each other.
once you make the drops, quickly shake the slide one time to remove excess liquid and put the slide on a slide warmer or hot plate at 70C for 10 minutes.

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