Zeiss Lightsheet Z.1, Руководство по эксплуатации

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CHAPTER 2 - SAMPLE PREPARATION
Carl Zeiss Sample Mounting for LSFM Lightsheet Z.1
24 000000-1790-528 02/2013
2.3.5 Hydrogel Preparation
Every gelling agent is prepared following specific protocols that vary widely from supplier to supplier, and
from laboratory to laboratory depending on the final application. We will not try to cover every single one
of these but rather give a simple protocol that we have been using in our laboratory to prepare
embedded samples in agarose as a gelling agent. The preparation is done as follows:
1.
Preparing a 1 % low melting agarose gel
Weigh 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. Swirl
to mix the solution.
Place the flask in the microwave. Heat above 95 °C until the solution is completely clear and no small
floating particles are visible. Do not allow the agarose to boil over as this will affect the final agarose
concentration. Swirl the flask frequently to mix the solution, prevent the agarose from burning, and
prevent 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 gel
Once molten the gel is left to cool to 37 °C (or just above gelling temp - read the material properties
sheet) in a water bath or on a heating plate. It is very important, especially for sensitive samples, to
ensure 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 a
heating block (80 °C – 90 °C) then transferred to a heating block at 37 °C.
3.
Using the gel
At 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. Work
quickly as the low melting point agarose will polymerize rapidly as it was kept at 37 °C close to the
gelling temperature.
4.
Polymerization of the gel
Let the gel polymerize. Avoid contact with any water-based solution as it will dilute the gelling agent
solution. The process of polymerization can be accelerated by cooling down the embedded sample (cold
water, fridge…) – but keep in mind that this might affect viability of a living sample.
5.
Using the prepared sample
Once fully polymerized, the embedded specimen can be manipulated, but keep in mind that it is a gel
and therefore fragile. Avoid any kind of friction, or shock etc. As it is a water-based object it must be
kept wet at all times to avoid drying out and damaging the sample. Moreover, many types of gel may
change 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 you
plan to reuse it.