Zeiss Lightsheet Z.1 Operating Manual

Contents

CHAPTER 2 - SAMPLE PREPARATIONCarl Zeiss Sample Mounting for LSFM Lightsheet Z.124 000000-1790-528 02/20132.3.5 Hydrogel PreparationEvery gelling agent is prepared following specific protocols that vary widely from supplier to supplier, andfrom laboratory to laboratory depending on the final application. We will not try to cover every single oneof these but rather give a simple protocol that we have been using in our laboratory to prepareembedded samples in agarose as a gelling agent. The preparation is done as follows:1. Preparing a 1 % low melting agarose gelWeigh 1 gram of low melting point ("low gelling") agarose ("Agarose Low Melt" (no 6351.1 from"http://www.carlroth.com") and place it in a flask. Add 100 ml of solvent (water, PBS) to the flask. Swirlto mix the solution.Place the flask in the microwave. Heat above 95 °C until the solution is completely clear and no smallfloating particles are visible. Do not allow the agarose to boil over as this will affect the final agaroseconcentration. Swirl the flask frequently to mix the solution, prevent the agarose from burning, andprevent boiling retardation.Wear heat-protective gloves when handling the flask.The agarose can be also sterilized for sterile use (cell culture).It is also possible to remove dissolved air bubbles using a vacuum pump.2. Cooling the gelOnce molten the gel is left to cool to 37 °C (or just above gelling temp - read the material propertiessheet) in a water bath or on a heating plate. It is very important, especially for sensitive samples, toensure that the agarose is at 37 °C before use.Note: Alternatively, you can aliquot your agarose solution into 1 ml or 2 ml Eppendorf tubes for later use.Label them and store them in a cool and dry place. In this case, each aliquot can be liquefied using aheating block (80 °C – 90 °C) then transferred to a heating block at 37 °C.3. Using the gelAt this stage follow the examples described in the section dealing with embedded sample preparation.Avoid bubble formation during handling and pipetting as they will impair the embedding process. Workquickly as the low melting point agarose will polymerize rapidly as it was kept at 37 °C close to thegelling temperature.4. Polymerization of the gelLet the gel polymerize. Avoid contact with any water-based solution as it will dilute the gelling agentsolution. The process of polymerization can be accelerated by cooling down the embedded sample (coldwater, fridge…) – but keep in mind that this might affect viability of a living sample.5. Using the prepared sampleOnce fully polymerized, the embedded specimen can be manipulated, but keep in mind that it is a geland therefore fragile. Avoid any kind of friction, or shock etc. As it is a water-based object it must bekept wet at all times to avoid drying out and damaging the sample. Moreover, many types of gel maychange their properties over time (e.g. swelling), and this can result in loosening of the gel in the support.It is therefore important to use your sample as soon as possible and monitor its quality over time if youplan to reuse it.