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CHAPTER 2 - SAMPLE PREPARATION
Lightsheet Z.1
Specific Examples of Sample Preparation
Carl Zeiss
02/2013
000000-1790-528
31
3.3
Preparation of a Fly Pupa (
Drosophila melanogaster
)
Some type of embryos are hydrophobic once dissected and cannot be mounted using the technique
described above as they will float on the agarose and will be impossible to embed. The following protocol
is suitable for this type of embryo such as fly embryos or pupas.
Equipment and reagents
−
Drosophila melanogaster
pupa or embryo
−
1 % Low Melting Point (LMP) Agarose in water or PBS
−
Capillary (Size 4, Blue, #701910, BRAND GmbH)
−
Heating block (90 °C and 40 °C)
Method
6.
Choose a pupa
Drosophila melanogaster
. Melt 1 % LMP agarose, aliquot 0.5 ml into a 1.5 ml
Eppendorf tube. Invert the tube to mix and allow agarose to cool to 40º C.
7.
To allow sample preparation the pupa must be submerged in agarose by pouring it directly on top
of it in a large drop of molten low melting point agarose.
8.
The pupa can then be pumped into a capillary as previously described.
9.
The insect can then be imaged.
Fig. 17
Mounting a
Drosophila
pupa.
(A) The pupa is prepared (labeling, drug treatment, dissected...) (B) The pupa is deposited in a watch
glass and covered by melted agarose to embed the hydrophobic pupa. (C) The pupa is then pumped
into the capillary. (D) The pupa can be imaged.
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