Dear all,
hi
I just had my first time cuting single cells with laser capture micro dissection microscope from Leica. The problem is that after cutting single cells from PEN membrane slides from iBD, the cut cell attach to membrane of the slide and do not fall to the eppendorf Tube. Anyone had this problem before? I am not drying my slides with ethanol as I got advice that too dry sample will have this attaching problem!
any experience?
Let et me know please
thank you in advance
The system works by cutting the membrane and the membrane makes up most of the weight, so that it falls according to gravity in the cap of the Eppendorf tube underneath the slide. Some of the problems, I've seen:
-make sure the tissue is on the membrane and not on the glass-only side of the slide: this will allow you to cut the membrane, but the tissue will never fall off...
-check if the you inverted the slide (gravity makes the the tissue and the membrane fall)
-make sure you went all around the cells, and that there is no connection anymore with the rest of the membrane
-I have seen people who didn't deparaffinize their slides, which makes it difficult for the tissue to come loose from
-Sometimes, there is water underneath the membrane, which creates a negative pressure underneath the slide... I don't really have a solution for that other than trying to dry it more.
-Sometimes there is static attachment of the membrane against the slide: try give a high-energy pulse that hits the loose fragment to make it fall. This might 'fry' the tissue and can result in worse quality DNA/RNA.
Hi,
Well, you can try to place a dehumidifier in the room and keep it running. You can also encourage the region to become unstuck by using the laser in manual control and just tapping on the region with the laser a few times. If that doesn't work, you may need to dissect smaller areas as these have less of a problem coming off.
Do you have access to a laser microdissection system from MMI? If yes, give it a try. MMI uses a patented CapLift technology to gently take up the sample by adhesive forces. During cutting, the adhesive isolation tube is in touch with the membrane (not with the sample directly) which will be cut together with the sample using a low-damage laser. Thus, you can't lose your sample!
If you don't have access to a MMI system, I guess I would try to cut larger areas which would increase gravity of your sample.
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