March 17, 2003

 

Instructions for Preparation of Fixative for TEM

 

Materials in kit:

  1. ampule:      10% glutaraldehyde, 10ml (store at 4C)
  2. ampule:      10% formaldehyde,10ml
  3. vial:               0.2M Na cacodylate buffer, 20 ml for fixative.
  4. vial:               0.1M Na cacodylate buffer, for washing.
  5. supplies: cup, razor blades, cutting board, plastic pipettes, extra sample vials.

 

Fixative preparation:

Break open ampules of aldehydes, add both to 0.2 M buffer. Final concentrations: 2.5% glutaraldehyde, 2.5% formaldehyde in 0.1M sodium cacodylate buffer (KarnovskyÕs fixative). Use within 24 hrs.

 

Method:

  1. Cover fresh tissue with droplet of fixative then mince with razor blades until segments have a minimum dimension of less than 1 mm. (i.e., 1 mm cube or 1 mm thick slices).
  2. Transfer tissue to a sample vial with at least 10 volumes of fixative
  3. Fix for 1 hour at room temperature then overnight at 4C.
  4. Replace fixative with 0.1 M cacodylate buffer and store at 4C (residual fixative retards microbial growth)

 

 

Ross Payne, rpayne@cvm.tamu.edu