The Huck Institutes of the Life Sciences

What does a general sample preparation protocol for electron microscopy look like?

Here are four examples of preparation protocols: for TEM and SEM of animal tissue, and for TEM and SEM of plant tissue.

Transmission electron microscopy (TEM) sample preparation

Animal tissue TEM preparation

  1. Primary Fixation: Immerse tissue blocks (usually 1mm3) in 2.5% glutaraldehyde in 0.1M Cacodylate buffer, pH 7.4 for 2 hours
  2. Primary Wash: 3 washes (each 5 minute duration) in 0.1 M cacodylate buffer with 4% sucrose, pH 7.4
  3. Secondary Fixation: Place sample in 1% osmium tetroxide in 0.1M cacodylate buffer, pH 7.4 for 1 hour
  4. Secondary Wash: 3 washes (each 5 minute duration) in 0.1 M cacodylate buffer, pH 7.4
  5. En Block Staining (optional): Stain samples with 1% uranyl acetate in 25% ethanol or water for 1 hour
  6. Dehydration: 1 x 10 min. in 50% ethanol1 x 10 min. in 70% ethanol1 x 10 min. in 90% ethanol1 x 10 min. in 95% ethanol3 x 10 min. in 100% ethanol (EM grade)3 x 10 min. in acetonitrile
  7. Infiltration and Embedding: Place sample in 50/50 acetonitrile/spurr’s resin for 1 hourPlace sample in 100% spurr’s resin for 1.5 hours – repeat once. Orient samples into mold and add resin
  8. Polymerization: Place mold containing samples in 60°C oven for 24 hours.

Plant tissue TEM preparation

  1. Primary Fixation: Immerse sample in 2.5% glutaraldehyde in 0.1M cacodylate buffer for 24 hours at 4oC
  2. Primary Wash: 3 washes (each 5 minute duration) in 0.1 M cacodylate buffer, pH 7.4
  3. Secondary Fixation: Place sample in 1% osmium tetroxide in 0.1M cacodylate buffer, pH 7.4 for 1 hour
  4. Secondary Wash: 3 washes (each 5 minute duration) in 0.1 M cacodylate buffer, pH 7.4
  5. En Block Staining (optional): Stain samples with 2% aqueous uranyl acetate for 1 hour or more.
  6. Dehydration: 2 x 10 min. in 50% ethanol2 x 10 min. in 70% ethanol2 x 10 min. in 90% ethanol2 x 10 min. in 95% ethanol3 x 10 min. in 100% ethanol (EM grade)3 x 10 min. in acetonitrile
  7. Infiltration: Place sample in 50/50 acetonitrile/spurr’s resin for several hours or overnight Place sample in 75/25 acetonitrile/spurr’s resin for several hours or overnightPlace sample in 100% spurr’s resin for several hours or overnight, repeat once.
  8. Polymerization: Place mold containing samples in 60°C oven for 24 hours.

Scanning electron microscopy (SEM) sample preparation

Animal tissue SEM preparation

  1. Primary Fixation: Immerse sample in 2.5% glutaraldehyde in 0.1M Cacodylate buffer, pH 7.4 for 2 hours at room temperature or at 4° C (in refrigerator) overnight.
  2. Primary Wash: 3 washes (each 5 minute duration) in 0.1 M cacodylate buffer pH 7.4
  3. Secondary Fixation: Immerse sample in 1% osmium tetroxide (aqueous) pH 7.4 for 1 hour at room temperature and in a light tight container.
  4. Secondary Wash: 3 washes (each 5 minute duration) in 0.1 M cacodylate buffer pH 7.4
  5. Dehydration: 1 x 10 min. in 25% ethanol1 x 10 min. in 50% ethanol1 x 10 min. in 70% ethanol1 x 10 min. in 85% ethanol1 x 10 min. in 95% ethanol2 x 10 min. in 100% ethanol1 x 10 min. in 100% ethanol (EM grade)
  6. Critical Point Dry: This automated process takes approximately 40 minutes to complete.
  7. Mounting: Mount the sample, which is now completely dried, onto metal stub with double sided carbon tape.
  8. Sputter Coating: Finally, apply a thin layer of metals (gold and palladium) over the sample using an automated sputter coater.  This process takes about 10 minutes.

Plant tissue SEM preparation

  1. Primary Fixation: Immerse sample in 2.8% glutaraldehyde in 0.1M Hepes buffer, pH 7.2 (with 0.02% Triton X-100), for several hours at room temperature or overnight at 4°C.
  2. Primary Wash: 3 washes (each 5 to 10 minute duration) in 0.1 M Hepes buffer, pH 7.2
  3. Dehydration: 1 x 10 min. in 25% ethanol1 x 10 min. in 50% ethanol1 x 10 min. in 70% ethanol1 x 10 min. in 85% ethanol1 x 10 min. in 95% ethanol2 x 10 min. in 100% ethanol1 x 10 min. in 100% ethanol (EM grade)
  4. Critical Point Dry: This automated process takes approximately 40 minutes to complete.
  5. Mounting: Mount the sample onto metal stub with double sided carbon tape.
  6. Sputter Coating: Finally, apply a thin layer of metals (gold and palladium) over the sample using an automated sputter coater.  This process takes about 10 minutes.