Materials & Methods
III Sample Preparation for Microscopy
The procedure of sample preparation for microscopy comprises four major steps: (1) fixation, (2) dehydration and embedding, (3) sectioning and (4) staining. For each step various alternatives and modifications are possible and the suitable variant is chosen depending on the respective experiment and intended mode of microscopy, sometimes also requiring combinations of two methods. The preparative techniques applied during this project are described in the following chapter. Table 3 gives an overview of the employed combinations.
Chemically fixed samples are either processed for resin embedding at room temperature, or plunge-frozen and sectioned in the cryostat. Physically or cryofixed specimen can also be transferred directly to the cryostat for sectioning or freeze substituted, followed by cryostat cutting or low-temperature embedding and subsequent ultramicrotomy. After suitable staining, cryostat sections and semithin resin sections can be investigated using a light microscope (LM) or confocal laser scanning microscopy (CLSM). Ultrathin resin sections are stained with heavy metal salts for transmission electron microscopy (TEM).
Table 3: Sample preparation and resulting microscopic applications.
Fixation Dehydration Embedding Sectioning Staining Microscopy
Histocytochemistry LM Chemical
sample fixation
At room temp. At room temp. Ultramicrotomy
Heavy metal staining TEM Chem. &
phys. fix. - - Cryostat Histocytochemistry
Immunofluorescence LM
- - Cryostat Histocytochemistry
Immunofluorescence LM CLSM
- Cryostat Immunofluorescence CLSM
Histocytochemistry CLSM Physical
sample
fixation Freeze
substitution Low-temp.
embedding Ultramicrotomy
Heavy metal staining TEM
tis-Materials & Methods sue, the biopsy needs to be fixed as soon as possible after its extraction. Fixation can be achieved either chemically or physically, depending on the demands of the respective method of investigation. Several methods for chemical as well as physical fixation are available, in-cluding the combination of both fixation forms.
III.1.1 Chemical Fixation
Glutaraldehyde and paraformaldehyde are the two most commonly used fixatives, either indi-vidually or in combination. Both aldehydes crosslink proteins or components of proteins.
Formaldehyde forms methylene bridges between two amino acids within a protein that are mostly reversible, while glutaraldehyde forms irreversible intramolecular and intermolecular protein crosslinks (Griffiths, 1993; Hayat, 2002). Uranyl acetate and osmium tetroxide are used as secondary fixatives after protein fixation, mostly combined with the first step of the embedding procedure. These heavy metals salts react primarily with lipid moieties.
Skin samples are cut into 2 mm pieces, in order to minimize the duration of the fixation, thus ensuring the penetration of the entire sample, and at the same time minimizing extracting effects of the fixative. The samples are usually treated with chemical fixatives for 24 h at 4 °C. Uranyl acetate is generally added to the primary fixative, while osmium tetroxide is applied for one hour prior to dehydration (see chapter III.2.1). Variations from this standard are described in the respective chapters. The applied fixatives and their preparation are listed below.
The samples are then either dehydrated and embedded in resins (see chapter III.2), plunge-frozen (see chapter III.1.2.1) or immediately sectioned with the cryostat (see chapter III.3.1).
Before plunge-freezing, the samples are infiltrated with sucrose solution after chemical fixa-tion for further 24 h at 4 °C, providing a protecfixa-tion against the formafixa-tion of ice crystals.
Paraformaldehyde stock solution 8% (w/v)
Paraformaldehyde (Plano, Wetzlar, Germany) 8.00 g
0.05 M HEPES buffer ad 100 ml
Hydrochloric acid ad pH 7.2
Paraformaldehyde (PFA) is dissolved with stirring at 60 °C. Sodium hydroxide solution is added drop by drop until the solution becomes clear, and the pH-value is adjusted to 7.2 with hydrochloric acid. The solution can be stored at -20 °C for several months.
Materials & Methods
Paraformaldehyde fixative (4 %)
PFA-stock solution 8 % (w/v) 50 ml
0.05 M HEPES buffer ad 100 ml
The solution can be stored at -20 °C for several months.
Fixative for immuno-electron microscopy (IEM-fixative)
PFA-stock solution 8 % (w/v) 50 ml
Glutaraldehyde solution 25 % (w/v) (Plano, Wetzlar, Germany) 40 µl
0.05 M HEPES buffer ad 100 ml
Hydrochloric acid ad pH 7.2
The solution should be mixed under inert gas (e.g. nitrogen), as glutaraldehyde is unstable in the presence of oxygen. The solution can be stored at -20 °C for several months.
Uranyl acetate 2% (w/v)
Uranyl acetate (SERVA, Heidelberg, Germany) 0.20 g
Aqua bidest. ad 10 ml
Uranyl acetate is applied at a concentration of 1 % for fixation. The solution can be stored at 4 °C for several weeks. Security guidelines for protection against radiation need to be considered when working with uranyl acetate.
Osmium tetroxide solution 1% (w/v)
Osmium tetroxide (Plano, Wetzlar, Germany) 1.00 g
Aqua bidest. 100 ml
Bidest. water is filled into a brown glass bottle with ground glass stopper. The ampoule with osmium tetroxide is cleaned on the surface with ethanol and put into the water-filled glass bottle. The ampoule is forced open with a pestle under water to prevent the release of osmium vapours. The osmium tetroxide is totally dissolved with stirring and filtered. The solution can be stored at 4 °C for several weeks, the refrigerator needs to be equipped with ventilation.
Sucrose solution 30% (w/v)
Sucrose (Merck, Darmstadt, Germany) 30.00 g
Aqua bidest. 100 ml
Materials & Methods III.1.2 Cryofixation
Cryofixation, or physical fixation, presents an alternative to chemical fixation that is not only faster but also eliminates side effects of chemical cross-linking, like the reorganization of the ultrastructure of cell membranes and cytoplasm. The term “cryofixation” describes the rapid cooling of a specimen to preserve a snapshot of its solution state. A major problem when it comes to cooling a specimen to subzero temperatures is the formation of ice crystals. If the ultrastructure of a sample is to be investigated, this must be avoided, as ice crystals seriously deform cell components (Echlin, 1992). As this can only be achieved by a very high cooling rate, samples should be chosen as small as is consistent with the physiological and morphological aims of the experiment. It is also possible to combine chemical and physical fixation methods to ensure stabilization against cryo-damage (B.M. Humbel and Müller, 1984).
III.1.2.1 Plunge-Freezing
The specimen is plunged into a suitable liquid cryogen as rapidly as possible. Propane or ethane are the most commonly used liquid cryogens. The sample size should not exceed 4 mm in diameter to ensure an efficient cooling rate. Sample and sample holder need to be config-ured to ensure that the sample is the first object to enter the cryogen.
Procedure
The cryogen, in this case liquid ethane, is contained in a metal reservoir and sitting in a bath of liquid nitrogen. The edge of the sample is held by a pair of tweezers that are clamped into a spring-loaded mechanical plunging device positioned above the cryogen bath. It is necessary to ensure that the injector is prevented from bouncing back and the sample does not hit the bottom of the cryogen container. The sample is injected into the cryogen with a velocity of approximately 5-6 m s-1. The average cooling rate for plunge-freezing is 10-12 · 103 K s-1. The sample is stored in liquid nitrogen until further preparation steps.
In this project, fresh skin biopsies without any pre-treatment and IEM-fixed, sucrose-infil-trated samples were preserved by plunge-freezing and processed for light microscopy, i.e.
cryostat sectioning with subsequent staining (see chapter IV).
Materials & Methods
III.1.2.2 High-Pressure Freezing
High-pressure freezing takes advantage of the fact that water can remain liquid well below its equilibrium freezing point without the formation of ice crystals. At atmospheric pressure this undercooling is possible until -38 °C. Below -130 °C water can be transferred into a stable non-crystalline condition, referred to as amorphous ice or vitrified water. In the temperature range between -38 °C and -130 °C nucleation, i.e. ice crystal formation, takes place. The application of high pressure (approx. 2.4 · 105 kPa) while cooling hinders the volume expan-sion associated with crystallization, and thus reduces the freezing point as well as the nucleation rate. Undercooling now continues until -92 °C without the interference of nucleation effects. This reduces the temperature (and time) range between the state of under-cooled and vitreous water during which nucleation occurs.
Although the cooling rate of this procedure (approx. 0.5 · 103 K s-1) is much lower than that of plunge-freezing, the reduction of cryoinjury of the sample makes it superior when it comes to investigating ultrastructure.
Procedure
Skin samples are taken using standard biopsy punches (diameter 2-4 mm). The subcutis is carefully removed and the sample is cut into small pieces of about 500-2000 µm in diameter and 200 µm in thickness. The samples are placed in standard aluminium platelets filled with incompressible 1-hexadecene to improve energy transfer and to protect the specimen. The samples are frozen in an HPM 010 high-pressure freezer (Bal-Tec AG, Balzers, Liechten-stein). Ethanol is used to apply the pressure (2.4 · 105 kPa) before cooling the specimen by a jet of liquid nitrogen.
The specimens are stored in liquid nitrogen until further processing.
III.1.2.3 Freeze-Drying
Frozen specimens can also by dehydrated by low-temperature vacuum sublimation. For total removal of residual water the samples are warmed to room temperature under high-vacuum.
Plunge-frozen samples of skin phototypes I-VI (see chapter II) were freeze-dried to facilitate transport to the Laboratory of Dr. Wakamatsu in Toyoake, Japan. The samples were stored in liquid nitrogen and transferred into the high-vacuum chamber of a BAF 060 Freeze-Fracture System (Bal-Tec AG, Balzers, Liechtenstein), equipped with a specimen stage, that can be
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.