Zeiss Lightsheet Z.1 Operating Manual

Contents

CHAPTER 2 - SAMPLE PREPARATIONLightsheet Z.1 Specific Examples of Sample Preparation Carl Zeiss02/2013 000000-1790-528 355. The pelleted cysts are permeabilized with PBS/1 % Triton X-100 for 10 minutes on a wheel orrocker 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 extraepitopes and efficiently mix the gel pellet.8. The gel pellet is incubated with the primary antibodies at the concentration indicated by thesupplier, 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 thesupplier, 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. Careshould 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 exoskeletonmade of chitin which is hydrophobic and autofluorescent. This characteristic allows imaging withoutlabeling by simply using the chitin autofluorescence to image the insect surface. This protocol applies toevery 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