19. Multiple places on the grid can be milled. When done, transfer
the shuttle back to the transfer unit, and close the valve.
20. After venting the airlock, place the transfer unit on the slush
chamber and pump. When the vacuum has reached 10
-2
, open
the valve on the transfer unit and place the sample in liquid
nitrogen. All steps in this part should be performed gently to
avoid any damage to the lamella. The slush chamber can be
pre-pumped beforehand so it can reach the desired vacuum
faster.
21. The sample can then be stored in clean liquid nitrogen for
cryo-ET.
2.3 Biological (Plant)
Tissues
The following will focus on plant tissues; however, the workflow of
High Pressure Freezing (HPF)—cryosectioning—cryo-ET can be
adapted to other types of tissues suitable for HPF. Preparation of
plant tissues for HPF and cryosectioning are discussed in further
detail in references [34, 35]. We will focus here on two plant tissues:
in vitro grown 6-day-old root tips and PSB-D Arabidopsis Landsberg erecta cells grown in liquid medium.
2.3.1 Tissue Cultivation
1. Liquid cultured PSB-D cells are grown in Murashig and
Skoog with Minimal Organics (MSMO, Sigma-Aldrich) with
sucrose pH 5.7 with added Kinetin (Sigma-Aldrich) and
1-Naphthaleneacetic acid (NAA) (Sigma-Aldrich). Dispatch
75 mL of media in four 250 mL flasks (Pyrex No 4980) and
cultivate at 25
C, continuous illumination (20 μE/m/s) with
120 rpm shaking.
2. Seeds are sterilized in bleach. 100 mL of commercial bleach is
placed in a beaker in a large plastic Tupperware, in a chemical
fumehood (see Note 12). Open 1.5 mL Eppendorf tubes containing the seeds are placed in a tube rack inside the plastic
Tupperware. 3 mL of 100% HCl are added and the Tupperware
is closed and left for 3–4 h. After treatment, the Tupperware is
opened, and tubes are closed before transferring to a sterile
hood. The seeds are aired out in the sterile hood by opening
the tubes for 30 min. The bleach-HCl mix can be left in the
chemical fumehood to evaporate.
3. Arabidopsis seedlings are grown in Murashig and Skoog
medium + vitamins (Duchefa Biochemie), with 2-(N-morpholino)-ethanesulfonic acid (MES) buffer (Euromedex) and 3.5 g
of plant agar (Duchefa Biochemie), pH 5.8 in square plastic petri
dishes (VWR).
4. Sterilized seeds are resuspended in autoclaved DI water, then
pipetted in horizontal lines onto the plates. The plates are then
set vertically in a greenhouse at 22
C, on a long day photoperiod (16 h, 100 μE/m/s) for a week to produce young
root tips.
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Mohammed Kaplan et al.
the shuttle back to the transfer unit, and close the valve.
20. After venting the airlock, place the transfer unit on the slush
chamber and pump. When the vacuum has reached 10
-2
, open
the valve on the transfer unit and place the sample in liquid
nitrogen. All steps in this part should be performed gently to
avoid any damage to the lamella. The slush chamber can be
pre-pumped beforehand so it can reach the desired vacuum
faster.
21. The sample can then be stored in clean liquid nitrogen for
cryo-ET.
2.3 Biological (Plant)
Tissues
The following will focus on plant tissues; however, the workflow of
High Pressure Freezing (HPF)—cryosectioning—cryo-ET can be
adapted to other types of tissues suitable for HPF. Preparation of
plant tissues for HPF and cryosectioning are discussed in further
detail in references [34, 35]. We will focus here on two plant tissues:
in vitro grown 6-day-old root tips and PSB-D Arabidopsis Landsberg erecta cells grown in liquid medium.
2.3.1 Tissue Cultivation
1. Liquid cultured PSB-D cells are grown in Murashig and
Skoog with Minimal Organics (MSMO, Sigma-Aldrich) with
sucrose pH 5.7 with added Kinetin (Sigma-Aldrich) and
1-Naphthaleneacetic acid (NAA) (Sigma-Aldrich). Dispatch
75 mL of media in four 250 mL flasks (Pyrex No 4980) and
cultivate at 25
C, continuous illumination (20 μE/m/s) with
120 rpm shaking.
2. Seeds are sterilized in bleach. 100 mL of commercial bleach is
placed in a beaker in a large plastic Tupperware, in a chemical
fumehood (see Note 12). Open 1.5 mL Eppendorf tubes containing the seeds are placed in a tube rack inside the plastic
Tupperware. 3 mL of 100% HCl are added and the Tupperware
is closed and left for 3–4 h. After treatment, the Tupperware is
opened, and tubes are closed before transferring to a sterile
hood. The seeds are aired out in the sterile hood by opening
the tubes for 30 min. The bleach-HCl mix can be left in the
chemical fumehood to evaporate.
3. Arabidopsis seedlings are grown in Murashig and Skoog
medium + vitamins (Duchefa Biochemie), with 2-(N-morpholino)-ethanesulfonic acid (MES) buffer (Euromedex) and 3.5 g
of plant agar (Duchefa Biochemie), pH 5.8 in square plastic petri
dishes (VWR).
4. Sterilized seeds are resuspended in autoclaved DI water, then
pipetted in horizontal lines onto the plates. The plates are then
set vertically in a greenhouse at 22
C, on a long day photoperiod (16 h, 100 μE/m/s) for a week to produce young
root tips.
92
Mohammed Kaplan et al.