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v2.0

by z mbstaff

Introduction

This tutorial will guide you through the process of cryo-fixation of adherent cells grown on 6 mm sapphire disks at the Center for Microscopy and Image Analysis, UZH, Zurich.

  1. Prepare your tools and parts for high-pressure freezing. In the cupboard above the machine you will find: the three-piece cartridge consiting of an upper cylinder, special middle plate 6 mm and lower cylinder
    • Prepare your tools and parts for high-pressure freezing. In the cupboard above the machine you will find:

    • the three-piece cartridge consiting of an upper cylinder, special middle plate 6 mm and lower cylinder

    • spacer rings 6 mm (200 μm)

    • carriers 0.1/0.2 mm

    • filter paper

    • mini-rack for middle plates

    • metal block

    • petri dishes

  2. In the cupboard on the main floor you will find: 2x HPF-tweezers 1x orange tweezer
    • In the cupboard on the main floor you will find:

    • 2x HPF-tweezers

    • 1x orange tweezer

    • LN2 sample storage box

    • box with 1-hexadecene Eppendorf tubes

    • screwdriver for LN2 sample storage box

    • EtOH glas beaker

    • heating plate

  3. Prepare all tools and accesories required for your cryofixation experiment. Check if the 200 µm spacer rings are completely flat otherwise replace them. Check if the 200 µm spacer rings are completely flat otherwise replace them.
    • Prepare all tools and accesories required for your cryofixation experiment.

    • Check if the 200 µm spacer rings are completely flat otherwise replace them.

    • Organize the required 100/200 cavities on a filter paper in a petri dish.

    • For 6 mm sapphire disks the 100 µm cavity must face the cells on the sapphire disk.

    • Insert the two-piece cartridge consisting of an upper cylinder and lower cylinder in the loading mechanism.

  4. Overview of sandwich configuration Overview of sandwich configuration
    • Overview of sandwich configuration

    • Visualization of sandwich configuration

    • Hold the sapphire at 45°.

    • Align the edge within the recess of the middle plate.

    • Release it.

    • Take a aluminium specimen type A (100/200 µm cavity) carrier with 100 µm cavity down and only wet this side with 1-hexadecane.

    • Add the 200 µm spacer ring on top.

    • Move the middle plate to the loading station.

    • Blot excess liquid away using filter paper.

    • Important here is that you blot fast and put some pressure to prevent shifting of the 200 µm spacer ring.

    • Flip the loading lever 180° until the process button is in position.

    • Initiate freezing by pressing the button.

    • After cryofixation, the two-piece cartridge and the specimen carriers are automatically ejected into the specimen dewar and are ready for transfer.

    • Open the sample dewar drawer and transfer the sample dewar to LN2 box.

    • Always pre-cool the tweezers prior to use.

    • Transfer all components including your sample to the LN2 box.

    • Always pre-cool the tweezers prior to use.

    • First, transfer the sapphire disk to the LN2 sample storage box.

    • Then transfer the specimen carriers to a beaker filled with 70% EtOH for cleaning.

    • Place the sample dewar back into the high-pressure freezer drawer.

  5. Place specimen carriers to a hot plate for drying.
    • Place specimen carriers to a hot plate for drying.

  6. Once all freezing is complete the two valves at the back can be opened. Allow LN2 to drain. Once all freezing is complete the two valves at the back can be opened. Allow LN2 to drain.
    • Once all freezing is complete the two valves at the back can be opened. Allow LN2 to drain.

  7. Once the storage dewar has reached a level of below 20% select SYSTEM form the main screen. Select AIR HEATER ON. This activates the heating cycle. The 80°C heating cycle will only start once the chamber temperature warms up to 10°C.
    • Once the storage dewar has reached a level of below 20% select SYSTEM form the main screen.

    • Select AIR HEATER ON.

    • This activates the heating cycle. The 80°C heating cycle will only start once the chamber temperature warms up to 10°C.

    • The bake out will automatically terminate at the end of the cycle leaving the instrument ready for the next user.

    • NOTE: Leave the main power to allow the heating cycle to run.

    • Once the air heating cycle is started, the compressor turns on "air heater working".

    • Once the cycle is complete the machine switches back to the standard screen and you can turn it off using the switch at the front.

Finish Line

z mbstaff

Member since: 05/06/2019

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