Arabidopsis thaliana(L.) Accession Col

Application Note for Leica EM AMW - Life Science Research

August 01, 2016

Arabidopsis thaliana (L.) accession Col-O
Plants were grown in growth chambers under defined conditions. After stratification for 4 days at 4°C seeds were grown in pots with soil with 9/15 hours day/night photoperiod. Day and night temperatures were 22°C and 18°C, respectively, the relative humidity was 60% and the plants were kept at 100% relative soil water content. Light intensity varied between 110 and 140 μmol m-2 s-1.

Six weeks after stratification the youngest fully developed rosette leaves were harvested two hours after the onset of the light period and prepared for electron microscopy with the Leica EM AMW.

Leaves at this stage were approximately 3.5cm in length and 1.5cm in width. Samples were taken from the middle of the leaves close to the middle vein. Small sections of leaves (1mm²) were cut with a razor blade on a modelling wax plate in a drop of 3% glutaraldehyde in 0.06M Sørensen phosphate buffer at pH 7.2.

Sections were then evacuated with a water jet vacuum pump for a maximum of 10 seconds in a vial filled with the above described fixative solution. Subsequently, the specimens were transferred into small baskets with a mesh width of about 200μm. These baskets were then stacked on top of each other and transferred into the mono-mode chamber of the Leica EM AMW which al­ready contained a vial filled with the above mentioned fixative solution. Microwave fixation was then started about 2 minutes after the cutting of the samples by starting the previously programmed protocol.

Sample preparation for transmission electron microscopy (TEM) was performed in order to develop a stan­dard protocol that would reduce sample preparation time for TEM-investigations. Therefore the overall and fine structure of leaf cells prepared with the Leica EM AMW were compared with leaf cells that were prepa­red with a conventional fixation protocol at room temperature. Additionally, the diameter of membranes from different cell compartments (chloroplasts, nuclei and plasmamembrane) was determined by using quantitati­ve computer supported transmission electron microscopy.

Epon, Agar 100
Standard mixture: Glauert Medium

Epon (Agar 100 Harz, Fa. Gröpl)20ml24g
DDSA (Dodecenylsuccinic adhydrid)16ml16g
MNA (Methylnadicanhydrid)8ml10g
BDMA (Benzyldimethylamin)1,3ml

1,2g


Epon soft:Epon hard:
Epon24gEpon24g
DDSA22gDDSA9g
MNA6gMNA15g
BDMA1,2gBDMA1,2g

 

 

Processing
vialsteptimemax. temp. (°C)reagentmodemax. power (W)
1100:02:0037Buffer + glutar aldehydeCont.15
1200:02:0020Buffer + glutar aldehydeCont.0
1300:02:0037Buffer + glutar aldehydeCont.15
1400:02:0020Buffer + glutar aldehydeCont.0
2100:00:4037BufferSlope20
3100:00:4037BufferPulse15
4100:00:4037BufferSlope20
5100:12:0037Buffer + OsO4Cont.15
6100:01:0037BufferCont.15
7100:01:0037BufferCont.15
8100:01:0037BufferCont.15
9100:01:0037Acetone 50%Slope20
10100:01:0037Acetone 75%Slope20
11100:01:0037Acetone 90%Slope20
12100:02:0037AcetoneSlope20
13100:02:0037AcetoneSlope20
14100:03:0037Resin 3:1Cont.10
15100:03:0040Resin 1:1Cont.10
16100:03:0045Resin 1:3Cont.10
17100:03:0050EponCont.12
18100:03:0050EponCont.12
19100:03:0050EponCont.12
Total time:00:49:20

 

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Fig.1: Overview
M = Mitochondria, N = Nucleus, CW = Cell Wall, V = Vacuoles
Fig.2: Detail 1
M = Mitochondria, N = Nucleus, CW = Cell Wall, V = Vacuoles
Fig.3: Detail 2
M = Mitochondria, N = Nucleus, CW = Cell Wall, V = Vacuoles

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