Procedure:

(A) Fixation : The samples for TEM are fixed by two different ways, (1) immersion or (2) perfusion. Fixation time, concentration of fixative agents depends on tissue thickness. It is performed in following steps:
1. Sample is incubated with 2% paraformaldehyde/2.5% glutaraldehyde in 50mM sodium cacodylate pH 7.4 for overnight at 4⁰C.
2. Post fixation, samples are incubated with 2% osmium tetraoxide in 50mM sodium cacodylate pH 7.4 for overnight at 4⁰C.

(B) Dehydration: Biological samples are fragile and contains large amount of water. Water present in the biological sample diffract electron rays and may increase the background signal. Following osmium fixation, water is chemically extracted from the specimen using a graded series of ethanol.
It is performed in the following steps:

• Sample is incubated with 35% ethanol for 15 mins.

• Remove the solution and  sample is incubated with 50% ethanol for 15 mins.

• Remove the solution and  sample is incubated with 70% ethanol for 30mins.  

• Remove the solution and  sample is incubated with 95% ethanol for 15 mins.  

• Remove the solution and  sample is incubated with 100% ethanol for 15 mins.  

• Remove the solution and  sample is incubated with 100% ethanol for additional 30mins.
• Remove the solution and  sample is incubated with propylene oxide for 15 mins.  
• Remove the solution and  sample is incubated with propylene oxide for additional 15 mins.  

(C) Embedding : A thin (~10nm-100nm) section of the sample so that electron can pass through it to form an image. Biological samples are fragile and can not be processed to cut thin section. Hence, sample needs to be embedded into a solid matrix to cut the sections. There are two different matrix used to embedded the sample for sectioning purposes:

(i) Epon embedding

• Remove the solution and  sample is incubated with propylene oxide: Epon resin (1:1) over night.
• Remove the solution and  sample is incubated with fresh Epon 812 resin  for additional 1-5 hrs.
• Dispense few µl fresh epon 812 resin in polyethylene capsules and specimen is transferred into it.
• Place capsule at 60⁰C in hot oven for 48hrs.

(ii) LR white embedding

•  Dehydrate the specimen in ethanol as described.
• Incubate tissue or cells for 30 min in mixture of Ethanol and LR white resin (1:1).
• Subsequently, incubate tissue or cells for 30 min in mixture of Ethanol and LR white resin (2:1).
• Incubate tissue or cells in LR white resin for 1hr. repeat this step 3 times.
• Finally leave the tissue or cells for overnight in 100% LR white resin.
• Fill the capsule with LR white containing tissue/cell and seal the capsule to allow polymerization of resin in 50⁰C oven overnight.