Interested in the detailed structure of your tissue? While it may be a rather complicated procedure with nasty chemicals, the advantages of epon embedding can make it the best choice for morphological studies. The hard blocks are excellent for structural preservation and the ability to section tissue thin enough for electron microscopy.
We previously introduced embedding in resin as an alternative to paraffin embedding. By the end of this article you will hopefully know what you need (and maybe some extra) to start embedding. A lot of variations exist for embedding protocols, so this article will stick to the most widely used options to walk through an example process.
Mind Your PPE’s
The embedding menu is loaded with highly toxic chemicals. The fixative properties of glutaraldehyde and osmium tetraoxide do not distinguish between the tissue in your dish and the tissue in your lungs. A fume hood is a must and keep the samples covered during each step to avoid rapid evaporation of the most volatile chemicals. Many of the other chemicals eat latex for lunch and even break through nitrile gloves relatively quickly, so wear proper gloves and replace them quickly if exposed.
Fix Your Tissue and Fix It Again
1. Primary fixation in 2.5% glutaraldehyde in phosphate buffer
Your primary tissue fixation step—though it occurs prior to the actual embedding process—is one of the most important. Without adequate structural preservation, the arduous task of embedding is a big waste of time when you finally get the tissue under the electron microscope. Use glutaraldehyde for the initial fixation—a solution of 2.5% glutaraldehyde in phosphate buffer is common. Perfusion fixation produces the best results, but if it’s not possible, immersion in glutaraldehyde for 2 hours to overnight (depending on the size of your tissue) works too.
2. Postfixation with 1% osmium tetraoxide
Osmium tetraoxide is the first of the embedding chemicals you should hope to never meet outside a lab. Though highly toxic, its ability to stabilize proteins and bind lipid molecules make it a valuable tool for electron microscopy. Osmium tetraoxide also darkens the surfaces it binds to, providing clear contrast of cellular structures under an electron beam.
Your sections will get extremely brittle after post-fixation, so carefully flatten and remove any folds before contact with osmium tetroxide. After this step, leave the tissue in the dishes and replace the solutions around it to prevent breaking your sections.
Keep Your Gloves On
3. Stain with 1% uranyl acetate in 70% ethanol
Uranyl acetate is a heavy metal. When it binds to proteins and lipids, its high electron density produces clear contrast of cell membranes. It is also highly toxic and mildly radioactive, making the bright yellow color look more sinister than cheery. Preparation of the solution in ethanol enhances solubility, allowing the uranyl acetate to penetrate the tissue faster.
4. Prepare the epoxy resin: Embed 812 + Araldite 502 + DDSA + BDMA
There are many different types of embedding media, but epoxy resin is the most widely used and best option for morphological studies. You can use either araldite resin or epon resin (Araldite 502 or Embed812 are both common). However, a combination of the two allows you to take advantage of the best qualities of each—thermal stability with araldite and high image contrast with epon.
Epoxy resin components are toxic and potentially carcinogenic (am I starting to sound redundant?) and are not safe until completely polymerized. If you want to do immunohistochemical studies on the tissue, epoxy resin can pose a challenge. Try staining the tissue before embedding, or check out other resins and additives that allow you to stain the tissue after embedding.
A downside of epoxy resin is the high viscosity of the components. Heat each one separately the night before to make them easier to measure and mix. Make sure you mix thoroughly to obtain properly hardened resin blocks. Combine the first three ingredients, mix well, but gently, to avoid bubbles. Then add the BDMA and mix well. Watch the solution turn a deep orange color. BDMA accelerates polymerization and slight variations in the concentration will affect the brittleness of the block. Another commonly used accelerant is DMP-30, though BDMA is often preferred because it has lower viscosity an is therefore easier to measure and mix thoroughly.
5. Resin infiltration via propylene oxide
Since araldite and epon are not reactive with alcohols, propylene oxide acts as a transition solvent between the alcohol dehydrations and epoxy resin. The graded process shifts from 100% propylene oxide to 100% resin. Propylene oxide is volatile, highly toxic, and a potential carcinogen. Remember your PPE? As an alternative, you can use acetone as a transition fluid.
What Went Wrong With the Embedding?
Errors in any of the steps alter the outcome of either the tissue preservation or resin block, which affects the quality of your tissue sample in the electron microscope. If you experience the unfortunate disappointment of making it through a long protocol to produce an unusable sample, check these common troubleshooting techniques below.
Your Resin Block Is Soft or Tacky
If your block isn’t hard enough, the likely culprit is mixing. If the components weren’t thoroughly mixed, the resin won’t harden properly. Soft resin also results from aggressive mixing that introduces air bubbles to the mixture. Gently mix the resin manually by inverting the tube to prevent the formation of air bubbles.
Brittle Resin Block
Slight changes in the amount of accelerator have drastic effects on the brittleness of the resin block. Double check the concentration recommended for your selected resin. You may need to make an adjustment to the amount of accelerator added.
Giant Needle-Like Crystals Obscure Your View of the Tissue in the Microscope
If you these, you are probably looking at precipitates of the uranyl acetate. Uranyl acetate is photosensitive and prepared as a saturated solution. Prepare it as needed and keep it protected from light. Perform the staining in the dark to avoid the formation of precipitates and be sure to clear any remaining uranyl acetate from the tissue when the staining is complete.
Uranyl acetate alone is often sufficient to produce the necessary contrast, but double staining with lead citrate achieves higher contrast if needed. As another heavy metal, it enhances contrast of cellular structures.
These are just some of the details you need to see the details in your samples. Do you have any tricks for epon embedding?
|Primary fixation||2.5% glutaraldehyde||2–24 h||Rinse in phosphate buffer|
|Post-fixation||1% osmium tetraoxide||1–2 h||Rinse in phosphate buffer|
|Dehydrations||50% EtOH||10 min||Repeat once|
|Dehydrations||70% EtOH||10 min||Repeat once|
|Contrast staining||1% uranyl acetate in 70% EtOH||1 h||Perform in the DARK!|
|Rinses||70% EtOH||5 min||Repeat once|
|Prepare epon resin||12.5 mL Embed 812|
7.5 mL Araldite 502
27 mL DDSA
1.3 mL BDMA
|Heat the components separately at 60C overnight before mixing|
|Dehydration||95% EtOH||5 min||Repeat 3 times|
|Dehydration||100% EtOH||5 min||Repeat 3 times|
|Dehydration||100% propylene oxide||5 min||Repeat 3 times|
|Resin infiltration||1:1 epon resin : propylene oxide|
1:2 epon resin : propylene oxide
100% epon resin
|Polymerization||72 h||Heat at 60'C|