Materials & Methods tempered from -170 °C to +50 °C. The vacuum chamber was pre-cooled to -140 °C and the vacuum-pressure was adjusted to 8 · 10-6 Pa. The temperature was gradually increased in steps of 30 °C every half hour. In a final step, the samples remained at -20 °C for one hour, before releasing them from vacuum and transfer to 1.5 ml Eppendorf tubes. The Eppendorf tubes were deposited in transport containers with anhydrous silica gel to ensure dry conditions during transport to Japan.
As the samples were disintegrated for chemical analysis, the unavoidable molecular or struc-tural damage brought about by shrinkage or collapse of the tissue during the desiccation process was no concern.
Materials & Methods
transferred into molds containing pure liquid resin and are finally placed into an oven (T = 60 °C) to polymerize the resin to form a solid block. The sample is positioned at the tip of the embedding container and aligned for sectioning later on, to achieve sections perpen-dicular to the skin surface and include all epidermal layers. The exact protocol of the proce-dure is displayed in Table 4.
It is of exceeding importance to keep the dehydration solvents sealed tightly, as they will ab-sorb water from the air to an extent that makes them incapable of eliminating water in the sample. Therefore, molecular sieves are used to desiccate the solvents. When replacing one dehydration agent with the next solution it is important to do so rapidly to avoid water uptake.
Any dehydration solvent is a powerful extractor of lipids. Osmium tetroxide or uranyl acetate can therefore be used during or after fixation to help stabilize the lipids during the dehydration process. Fixation steps are usually followed by washing with HEPES buffer.
Table 4: Protocol for room temperature dehydration and embedding.
Step Media Duration Temperature
HEPES buffer, pH 7.4 2 x 10 min room temp.
Osmium tetroxide 1 %* 1 h room temp.
HEPES buffer, pH 7.4 * 2 x 10 min room temp.
30 % ethanol 15 min room temp.
50 % ethanol 15 min room temp.
70 % ethanol 30 min room temp.
90 % ethanol 30 min room temp.
100 % ethanol 1 h room temp.
dehydration
100 % ethanol 1 h room temp.
30 % Epon in ethanol 1 h room temp.
50 % Epon in ethanol 1 h room temp.
70 % Epon in ethanol 1 h room temp.
90 % Epon in ethanol 1 h room temp.
100 % Epon over night room temp.
100 % Epon 2 h room temp.
embedding
100 % Epon 72 h 60 °C
* not obligatory
Materials & Methods Epon (Fluka, Munich, Germany)
Stock solution I
Epoxy embedding medium 62 g
Hardener DDSA (dodecenyl succinic anhydride) 100 g
Stock solution II
Epoxy embedding medium 100 g
Hardener NMA (nadic methyl anhydride) 89 g
Epon
Stock solution I 40 g
Stock solution II 60 g
Accelerator DMP 30 (2,4,6-tridimethylamino methyl phenol) 1 g Epon should be freshly prepared for final embedding and polymerization steps. Excess Epon can be stored at -20 °C for several weeks and used for initial infiltration steps with low epoxy-ethanol ratio. The stock solutions can be stored at 4 °C for several months. Heating should be avoided while thoroughly mixing the stock solutions to prepare embedding medium.
III.2.2 Freeze Substitution and Low-Temperature Embedding
High-pressure frozen samples are ideally dehydrated below the recrystallization temperature of -130 °C, but this would be far too slow and only few solvents are able to dissolve ice at these temperatures. But recrystallization usually does not occur in hydrated specimens until approximately -80 °C and several solvents are available that dissolve ice in this temperature range in reasonable time. Anhydrous acetone is most commonly used as substitution medium (B.M. Humbel and Müller, 1984). For this project, acetone saturated with uranyl acetate and fluorescent dyes was applied.
Prior to freeze-substitution, excess 1-hexadecene is cautiously removed from the specimen under liquid nitrogen conditions and the sample is removed from the aluminium platelet. The samples are transferred into 1.5 ml Eppendorf tubes filled with precooled substitution me-dium, contained in transfer vessels (Hohenberg et al., 1994). The freeze substitution is per-formed in an Leica automatic freeze substitution system EM AFS (Leica, Bensheim, Ger-many), leaving the samples in the dehydration medium for 40 h at -90 °C. Once the
substi-Materials & Methods
tution is completed, the specimens are allowed to slowly warm to -50 °C over a period of 20-24 h and washed twice with acetone and subsequently with ethanol until excess fluorescent stain is removed. The exact protocol is listed in Table 5. Subsequently the samples are infil-trated with low-temperature embedding resin (Lowicryl® HM20; Polysciences, Eppelheim, Germany). Concentrations of 30 % and 70 % of HM20 in ethanol are applied twice, each time for one hour, followed by pure HM20 over night. Prior to UV polymerization fresh HM20 is applied and the resin is cured at -35 °C, followed by further polymerization at room temperature.
Table 5: Protocol for freeze-substitution and low temperature embedding.
Step Media Duration Temperature
UAc & fluorescent stains (1)
in acetone 40 h -90 °C
no change (2) 12 h -70 °C
no change 8-12 h -50 °C
acetone 1 h -50 °C
acetone 1 h -50 °C
ethanol 1 h -50 °C
ethanol 1 h -50 °C
ethanol 1 h -50 °C
dehydration
ethanol 1 h -50 °C
30 % HM20 in ethanol 1 h -35 °C
30 % HM20 in ethanol 1 h -35 °C
70 % HM20 in ethanol 1 h -35 °C
70 % HM20 in ethanol 1 h -35 °C
HM20 15-18 h -35 °C
HM20 2 h -35 °C
HM20 & UV polymerization 72 h -35 °C
embedding
UV polymerization 72 h room temp.
(1) Nile blue sulphate (Merck, Darmstadt, Germany) & DiIC18 (Molecular Probes, Leiden, the Netherlands), or Safranine O (Merck, Darmstadt, Germany)
(2) if the specimen are still held in HPF-platelets, they are carefully loosened without removing them from the substitution medium
Materials & Methods Lowicryl® HM20
Crosslinker D 2.98 g
Monomer E 17.02 g
Initiator C 0.10 g
Crosslinker and monomer are mixed gently by slowly rocking the vial from side to side. The formation of air bubbles or foaming needs to be avoided, as incorporation of oxygen impairs with polymerization. HM20 should be freshly prepared for final infiltration steps polymeri-zation. Excess HM20 can be stored at -20 °C for several weeks and used for low concen-tration infilconcen-tration steps. Polymerization should be tested prior to application.