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CHAPTER 2 - SAMPLE PREPARATION
Lightsheet Z.1
Specific Examples of Sample Preparation
Carl Zeiss
02/2013
000000-1790-528
35
5.
The pelleted cysts are permeabilized with PBS/1 % Triton X-100 for 10 minutes on a wheel or
rocker to efficiently mix the gel pellet.
6.
The gel pellet is washed twice with PBS (500-1000 g, 5 minutes).
7.
The gel is incubated for 10 minutes in PBS/1 % FCS on a wheel or a rocker to block the extra
epitopes and efficiently mix the gel pellet.
8.
The gel pellet is incubated with the primary antibodies at the concentration indicated by the
supplier, using a wheel or rocker to efficiently mix the gel pellet.
9.
The gel pellet is washed twice with PBS (500-1000g, 5 minutes).
10.
The gel pellet is incubated with the secondary antibodies at the concentration indicated by the
supplier, using a wheel or a rocker to efficiently mix the gel pellet.
11.
The gel pellet is washed twice with PBS (500-1000 rpm, 5 minutes).
12.
The cysts can be stained at this stage with Hoechst to label the nuclei.
13.
The gel is pelleted and as much of the supernatant as possible is removed.
14.
The gel pellet is mixed with low melting point agarose, mixed and pumped into a capillary.
The extracellular gel tends to clump once fixed and may stay as one piece when mounting. Care
should be taken to quickly but efficiently resuspend the gel.
15.
Allow the agarose to polymerize.
16.
Fill the chamber with PBS prior to introducing the sample for imaging.
3.7
Preparation of a Whole Mount of a Mosquito (
Anopheles gambiae
)
Adult insects are very large objects that can be up to 5 cm long. Moreover, they have an exoskeleton
made of chitin which is hydrophobic and autofluorescent. This characteristic allows imaging without
labeling by simply using the chitin autofluorescence to image the insect surface. This protocol applies to
every type of adult insects as well as for various type of plankton.
Equipment and reagents:
−
An adult
Anopheles gambiae
−
1.5 % Low Melting Point (LMP) agarose in water or PBS
−
1 ml syringe (BD Biosciences)
−
Ethanol (70 %)
−
Glycerol (50 %)
−
Sucrose
−
Heating block
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