Zeiss Lightsheet Z.1, Руководство по эксплуатации

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CHAPTER 2 - SAMPLE PREPARATION
Carl Zeiss Specific Examples of Sample Preparation Lightsheet Z.1
30 000000-1790-528 02/2013
1. Select embryos for imaging, dechorionate. Melt 1.5 % LMP agarose, aliquot 0.5 ml into a 1.5 ml
Eppendorf tube. Add 150 ml of Mesab to ensure that the embryos do not move during imaging.
Invert the tube to mix and allow agarose to cool to 40º C.
2. Add the embryo to the tube containing agarose using a Pasteur pipette, transferring as little buffer
as possible.
Add the embryo to the Eppendorf tube as a drop on the tube wall. If necessary remove the extra
buffer with a yellow tip before dropping the embryo into the agarose.
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Or transfer the embryo to an empty Eppendorf tube, remove all medium and add the liquid
agarose.
3. Let the embryo fall to bottom of the Eppendorf tube. Insert a capillary into the tube and suck the
embryo into it by pulling out the thread or plunger like a syringe piston.
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When sucking up the agarose, make sure that initially the plunger is sticking out of the capillary
within the liquid agarose, to avoid air bubble formation. Furthermore leave some space between
the plunger and the sample (see Fig. 16/
D )
4. Allow the agarose to harden and place the capillary in a stand in water or PBS.
5. Mount on the capillary sample holder prior to imaging.
Fig. 16
Mounting an Oryza latypes embryo. (A) The embryo is prepared (labelling, drug treatment,
dissected...) (B) The embryo is deposited on the side of the Eppendorf tube and the excess of
water is removed with a pipette. (C) The embryo is dropped into the agarose and pumped into the
capillary. (D) The embryo can be imaged.