# Embedding in Resin ## Embedding in Resin ![Zebrafish Tail Muscle](/sites/g/files/omnuum4896/files/styles/hwp_21_9__1920x825/public/2024-11/Screenshot%202024-11-18%20at%209.50.26%20AM.png?itok=z2STvWs2) ## expand\_more The routine method for examining biological samples in TEM is to embed the material in plastic and cut ultrathin (60-80nm) sections. The choice of resin depends on the type of sample and the purpose of your study. Listed below are some of the more commonly used resins. ### **Epoxy resins** **Epon or Epon-Araldite** mixtures are the most widely used resins for electron microscopy. Epon is excellent for morphological studies but not a good choice for most immunocytochemistry purposes. The sample has to be completely dehydrated in protein denaturing solvents (like propylene oxide or acetone) before infiltration, and polymerization has to take place at temperatures around 50-60° C. **Spurr’s Resin** is a Low Viscosity mixture which provides rapid infiltration of tissues. We use it for bacteria, yeast, parasites and skin tissues that are difficult to infiltrate well with Epon. It's easy to prepare and mixes rapidly. Spurr's resin is compatible with ethanol so no change to propylene oxide is needed prior to infiltration, although we recommend it. Polymerization at 60°C is recommended. ### **Routine Protocol for Embedding in Resin** Our standard fixative is a mixture of 1.25% formaldehyde, 2.5 % glutaraldehyde and 0.03% picric acid in 0.1 M Sodium cacodylate buffer, pH 7.4. We provide a 2x solution. You can also use a commercially available mixture of 2.5% glutaraldehyde and 2% formaldehyde in 0.1M Sodium cacodylate buffer, pH 7.4. **Fixation of cells:** Add fixative (2x concentration) 1:1 to the cell media. Leave for 1 hour at RT. If cells grow in suspension, spin them down in the fixative at 3000rpm for 3 minutes. Leave for 1 hour at RT in the fixative. **Fixation of tissue:** Perfusion fixation is preferred. For this we recommend using 2% formaldehyde and 2.5 % glutaraldehyde in 0.1 M Sodium Cacodylate buffer, pH 7.4. After perfusion, dissect out the tissue of interest, immerse in fresh fixative and cut into 1-2 mm cubes - leave to fix at least 2 hours at RT. ***Bring your fixed samples to the EM lab in the fixative at this point.*** We will follow the protocol below: Wash in cacodylate buffer 3x 1% Osmiumtetroxide/1.5% Potassiumferrocyanide (in H2O) leave for 1 hour at RT Wash in H2O (or in malelate buffer pH 5.15) 3x 1% Uranyl Acetate in H2O (or in maleate buffer) for 30 minutes - 1 hr Wash in H2O 3x Dehydration: 70% EtOH 15 min/ 90% EtOH 15 min/ 100% EtOH 2x15 min Propyleneoxide 1 hour Infiltration: Epon mixed 1:1 with propyleneoxide ON at 4 C Move samples to embedding mold filled with freshly mixed Epon Allow sample to sink, move to oven for polymerization Leave to polymerize for 24-48 hours at 60°C ### **Osmium Free Method for Resin embedding** (Tannic Acid/Uranyl Acetate method) 1\. Fix in PFA/ Picric acid as above. For immuno EM chose a fixative that works well for your antigen. (4% formaldehyde+0.01-0.1% glutaraldehyde is a good start) Do the following processing on ice until 100% Ethanol: 2\. Freshly made 1% Tannic Acid in Maleate Buffer (MB) for 40 min, then rinse 2x in MB 3\. In the dark: 1% Uranyl Acetate in MB for 40 min, then rinse twice in MB 4\. Dehydrate 5 min each: 50%/70% Ethanol 5\. 1% PPD(p-phenylenediamine) in 70% Ethanol for 15 min 6\. Rinse twice in 70% Ethanol 7\. Dehydrate 5 min each: 80%/95%/2x100% Ethanol 8\. Resin infiltration (LR white or other hydrophilic resin) 9\. Polymerization at 55-60° C for 2-3 hrs. ### **Acrylic Resins** Sort**LR White and LR Gold** are blends of hydrophilic and acrylic monomers that rapidly penetrate tissue because of their low viscosity. LR Gold is cured by UV-light in the cold while LR White can be cold cured (using an accelerator) or heat cured. The hydrophilic nature of these resins makes them usable for immunocytochemistry. **Lowicryl** K4M, HM20, K11M and HM23 are highly cross-linked acrylate and methacrylate based embedding media designed for use over a wide range of embedding conditions. They are usable for embedding at low temperatures and for immunocytochemistry.K4M -23°C hydrophilic HM20 -70°C hydrophobic K11M -60°C hydrophilic HM23 -80°C hydrophobic --- Attachments- [ picture\_as\_pdf Embedding Tissue Perfusion.pdf ](/sites/g/files/omnuum4896/files/2024-11/Embedding%20Tissue%20Perfusion_0.pdf) - [ picture\_as\_pdf Embedding Tissue Drop Fix.pdf ](/sites/g/files/omnuum4896/files/2024-11/Embedding%20Tissue%20Drop%20Fix_0.pdf) - [ picture\_as\_pdf Embedding Pellet.pdf ](/sites/g/files/omnuum4896/files/2024-11/Embedding%20Pellet_1.pdf) - [ picture\_as\_pdf Embedding Monolayers.pdf ](/sites/g/files/omnuum4896/files/2024-11/Embedding%20Monolayers_1.pdf) - [ picture\_as\_pdf Embedding Coverslips.pdf ](/sites/g/files/omnuum4896/files/2024-11/Embedding%20Coverslips_1.pdf) ---