Lowicryl® Resin Kits for Immunogold Staining
Product information sheet
Lowicryl® Low Temperature Embedding Resins
Introduction:
Lowicryl K4M, HM20, K11M, and HM23 are highly crosslinked acrylate and
methacrylate based embedding media which have been designated for use over a
wide range of embedding conditions.
See references 1, 2, and 3. These resins have been formulated to provide low viscosity
at low temperatures: K4M is usable to -35°C (-31°F), and HM20 to -70°C (-94°F).
K11M and HM23 have been developed with freezing points that allow applications down to
about -60°C and -80°C, respectively. All Lowicryl media exhibit low viscosity at temperatures
as low as -35°C (-31°F). The investigator also has a choice of either polar (hydrophilic: K4M
and K11M) or nonpolar (hydrophobic: HM20 and HM23) embedding media.
Lowicryl resins are photopolymerized by long wavelength (360 nm) ultraviolet (UV) light. Since the
initiation of the photopolymerization is largely independent of temperature blocks may be polymerized
at the same temperatures which are used for infiltration. A rapid polymerization method
has also been reported. See reference 4.
Proteins and lipoproteins are denatured in organic, water-miscible fluids
which are involved in conventional embedding techniques.
The hydrophilic properties of K4M and K11M provide two distinct advantages.
During dehydration and infiltration, the specimens may be kept in a
partially hydrated state since K4M and K11M may be polymerized with up to 5%
(by weight) water in the block.
Secondly, K4M and K11M are particularly useful for immunolabelling of
sections using specific antisera, lectins, and colloidal gold particles.
See references 5, 6, 7, and 8. The use of K4M or K11M
results in better structural preservation (See reference 9),
improved preservation of antigenicity and significantly lower background labeling
(See references 10, 11, 12, 13, 14, 15).
HM20 or HM23 can be used to produce high contrast images of completely unstained thin sections in the STEM
mode by atomic number contrast. See reference 16. HM20
and HM23 are particularly suitable for dark-field observation because of their low density, as compared to
conventional embedding media. They can also be used routinely at temperatures as low as - 70°C (-94°F). At
these low temperatures, biological material is stabilized and may even retain its bound water conditions.
All Lowicryl resins can be used for freeze-substituted samples.
See reference 17.
Preparation of Lowicryl Media:
The Lowicryls are provided in a highly purified 3-component system:
a) crosslinker
b) monomer mixture
c) initiator
The resin is stabilized, but it is not necessary to remove the stabilizer before use. By varying the
ratio of monomer to crosslinker, one can easily tailor the resin hardness to the needs of a particular
specimen, the more the crosslinker used, the harder the blocks.
For example:
- For HM20, the crosslinker concentration may be varied from 5-17 Wt% (1.0 to 3.5g/20g resin).
- For K4M, the crosslinker concentration may be varied from 4 to 18 Wt% (0.8 to 3.6 g/20g resin).
The resin should be prepared in a brown glass container or otherwise protected from direct light.
All of the components are readily miscible with each other. Avoid excessive stirring which may
result in the incorporation of oxygen into the resin, thereby interfering with the polymerization.
Gentle stirring with a glass rod for 3-5 minutes, or mixing with a stream of dry nitrogen gas bubbled
through the resin is recommended.
The mixtures below give blocks with about medium hardness to UV polymerization:
|
K4M | HM20 | K11M | HM23 2 |
|
-------- | -------- | -------- | -------- |
|
Crosslinker A 2.70g | Crosslinker D 2.98g | Crosslinker 1.0g | Crosslinker 1.1g |
|
Monomer B 17.30g | Monomer E 17.02g | Monomer 19.0g | Monomer 18.9g |
|
Initiator C 1 0.10 g | Initiator C 1 0.10g | Initiator 0.1g | Initiator 0.1g |
1 For polymerization of K4M or HM20 between -50°C (-58°F) to 0°C (-32°F). Above 0°C, the initiator
can be replaced by the same amount of benzoin ethyl ether. Room temperature polymerized blocks can be ready for
sectioning after only a few hours.
2 Above -50°C (-58°F), benzoin monomethyl ether and below -50°C (-58°F), then Igracure 651.
Any of the standard aldehyde fixation procedures may be used. Since the resins are in most cases photopolymerized,
the use of fixatives which also have staining properties (e.g.
osmium tetroxide) is not generally recommended (See reference 18).
A representative dehydration infiltration schedule for ethanol, which may be used for Lowicryl resins, is given
as follows:
|
Ethanol, Volume % | Temperature, °C | Time, minutes |
|
------------------------- | ---------------------- | ------------------ |
|
30 | 0 | 30 |
|
50 | -20 | 60 |
|
70 | -35 (-50) 3 | 60 |
|
95 | -35 (-50 to -80) 3 | 60 |
|
100 | -35 (-50 to -80) 3 | 60 |
|
100 | -35 (-50 to -80) 3 | 60 |
|
Resin: Ethanol, vol:vol | Temperature, °C | Time, minutes |
|
----------------------------- | ---------------------- | ------------------ |
|
1:1 | -35 (-50 to -80) 3 | 60 |
|
2:1 | -35 (-50 to -80) 3 | 60 |
|
100% resin | -35 (-50 to -80) 3 | 60 |
|
100% resin | -35 (-50 to -80) 3 | Overnight or4-16 hours |
3 Suggested steps for work at lower temperature, possibly only with HM20
(-70°C), HM23 (-80°C) and K11M (-60°C).
At all temperatures below 0°C. care must be taken not to allow the residual
water in the specimen be freeze during the dehydration step. A variety of
schemes can be developed for any other temperature or polar dehydrating
agent, as long as solubility allows it, including freeze substitution methods.
The biological consequences of using even lower temperatures needs to be explored. Nothing
precise is known ,as yet, about the completeness of dehydration at these low temperatures.
Lack of success, due to a loss of water, e.g. in the pyramid of the block, should not automatically
be interpreted as a "bad infiltration". The demonstrated persistence of lipids at lower temperatures
seems not to cause problems with infiltration. See reference 19.
A rapid method of fixation and embedding that avoids low temperatures using HM20 has also been reported,
see reference 20.
Polymerization:
Resins are polymerized by indirect long wavelength UV irradiation at 360 nm generally using two 15 watt
(FEI/Philips TLAD 15W/05 or equivalent) source at - 30°C to -40°C at a distance of 30-40 cm for 24 hours.
Hardening can be done in filled-to-capacity gelatin capsules,
BEEM® capsules, or the
SPI UV transparent silicone flat embedding molds.
Slow polymerization, particularly using diffuse radiation, produces superior blocks without severe shrinkage effects.
Sectionable preparations can be produced in as little time as 12 hours, though the sectioning quality improves when
they are further hardened under UV light at room temperature for 2-3 days.
Sectioning and staining:
Lowicryl easily yields silver to gray sections on diamond
or glass knives. Optimal staining can be done easily with
uranyl acetate and Reynold's lead citrate.
Oxidizing heavy metal stains like osmium tetroxide
(OsO4) or potassium permanganate (KMnO4)
produce inferior results due to reaction with the resin.
Osmium Ammine B at low pH shows a strong specificity for nucleic acids
(See reference 21).
Safety and Storage:
Be sure to examine closely the MSDS for these resin products before using. Always use protective gloves
when handling these materials such as the
SPI Supplies® Brand vinyl gloves.
To Ask a Question or Make a Comment
To Place an Order or Request a Quote
Return to:
Thursday February 09, 2012
© Copyright 2012. By Structure Probe, Inc.
Contacting SPI Supplies and Structure Probe, Inc.
All rights reserved.
All trademarks and trade names are the property of their respective owners.
Privacy Policy
Worldwide Distributors, Representatives, and Agents
