15 Preparation of Samples for CGH by DOP-PCR from microdissected cells
189
I Subprotocol 1
Microdissection
-1~ \" Procedure
Preparation of needles for microdissection
1. Place the low melt borosilicate glass sticks into the pipette puller.
2. Pull a sufficient number of glass needles for the experiment planned.
3. Discard each needle after using.
Note: The pipette puller settings will need to be optimized for any given
instrument. The tips are required to be short and sharp for cutting out
single cells from tissue sections.
Microdissection of cells from tissue sections
1. Wash 1O-llm-thick routinely fixed (4% neutral buffered formaldehyde)
paraffin-embedded tissue sections twice in xylol for 10 min.
2. Rehydrate the sections in a series of 50%-70%-90%-96% ethanol for
2 min each.
3. Wash 1 min in aqua dest.
Note: Non-neutral formaldehyde or long-term storage of the specimens in formaldehyde during the fixation results in a poor preservation of DNA which is not suitable for an efficient homogeneous amplification with DOP-PCR.
1. Stain the slides in Mayer's hemalaun (dilution 1:5) for 1 min.
2. Rinse the slides briefly with aqua dest.
3. Incubate the slides for 5 min in tap water at 37°C.
4. Rinse the slides briefly with aqua dest.
5. Transfer the slides into a Coplin jar containing 40 ml phosphate buffered saline (PBS, pH 7.4) and 1% glycerol.
Note: This step achieves an appropriately sticky consistency of the tissue for the following procedure.
Deparaffinization of
paraffin-embedded
tissue sections
Nuclear staining
of cryofixed or
paraffin-embedded
tissue sections
189
I Subprotocol 1
Microdissection
-1~ \" Procedure
Preparation of needles for microdissection
1. Place the low melt borosilicate glass sticks into the pipette puller.
2. Pull a sufficient number of glass needles for the experiment planned.
3. Discard each needle after using.
Note: The pipette puller settings will need to be optimized for any given
instrument. The tips are required to be short and sharp for cutting out
single cells from tissue sections.
Microdissection of cells from tissue sections
1. Wash 1O-llm-thick routinely fixed (4% neutral buffered formaldehyde)
paraffin-embedded tissue sections twice in xylol for 10 min.
2. Rehydrate the sections in a series of 50%-70%-90%-96% ethanol for
2 min each.
3. Wash 1 min in aqua dest.
Note: Non-neutral formaldehyde or long-term storage of the specimens in formaldehyde during the fixation results in a poor preservation of DNA which is not suitable for an efficient homogeneous amplification with DOP-PCR.
1. Stain the slides in Mayer's hemalaun (dilution 1:5) for 1 min.
2. Rinse the slides briefly with aqua dest.
3. Incubate the slides for 5 min in tap water at 37°C.
4. Rinse the slides briefly with aqua dest.
5. Transfer the slides into a Coplin jar containing 40 ml phosphate buffered saline (PBS, pH 7.4) and 1% glycerol.
Note: This step achieves an appropriately sticky consistency of the tissue for the following procedure.
Deparaffinization of
paraffin-embedded
tissue sections
Nuclear staining
of cryofixed or
paraffin-embedded
tissue sections