tissue ChIP - (Apr/19/2005 )
Hi, all ChIP experts / enthusiasts,
Could anybody like to share your experience / protocol on tissue ChIP?
The size of sections* of tissue sample : ca. 100 micron x 60 micron X 1 mm
a. could I make the fixation directly on it (2% formaldehyde, 30 min,RT ) ,
then sonicate it to dismember it as well shear the DNAs to 300-1000bp?
*I worry about such fixation can work well on this size sample (i.e. if formaldehyde can permeate well); but in some publications the fixation directly on tissue were applied ,
but no info about size of sections of samples.
Others performed fixation after pellet cell.
b. or should I step -by- steply dissoaciate the tissue as primary cell culture , then filter them using cheesecloth , then perform the fixation , lyse them by cell lysis buffer and isolate the nuclei ,etc?
2) May I use the Misonix sonicator 3000 to disaggregate and homogenize
the tissue ( or i have to use a Dounce homogenizer), then collect cell pellet ?
* I tried Dounce homogenizer but am not sure if Dounce homogenizer is helpful for homogenization on the sections of my tissue sample (100 micron x 60 micron x 1 mm).
How you check the homogenization? why some prefer the loose fitting A pestle
I am new in this field. I am also working on tissues and I have the same question. But from all the protocol's I see, It is no need to homogenize the tissue before fixation. One protocol said that tissues was cut into 4 mm^3 still works well.
Normal tissues, yes, as you mentioned: tissues was cut into 4 mm^3 still works well.
However, my sample tissue has the cuticle, so I worry about the fixation.
i tried collengase/trypsin digestion before fixation, such digestion cannot work on it.
i'm tryiong to find out solution on it.
anyway, thanks for your reply.