Routine solutions we use in our laboratory
Mammalian "Ringer's" solution:

(for maintaining vertebrate cells in a healthy, living condition during light microscopy or during any physiological or pharmacological manipulation(s) in preparation for EM:

(155 mM NaCl, 3 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 3 mM NaH2PO4, and 5 mM HEPES brought to pH 7.4 with NaOH, plus 10mM glucose.)

In making this up this solution, be sure not to add the CaCl2 until after pH'ing the whole mixture of other salts to 7.4, or else the calcium will precipitate when the NaOH hits the surface of the solution. (We usually add the CaCl2 from an unbuffered 1M stock in distilled water.)

For long term light microscopic recording of living cells we commonly add 1% BSA to this solution, so long as it will not adversly affect any drug-tratments we are trying (e.g., complex the drugs or something), or we even add 10% fetal calf serum (minus neutral red pH-indictor), if we want to keep physiological functions "revved up" to the max.

For fixing "broken" cells, we suggest using:

2% glutaraldehyde in what we call "KHMgE" buffer. This consists of 30 mM HEPES buffer brought to pH 7.4 with KOH (not NaOH!), with 70 mM KCl present to make it roughly isotonic to the intracellular milieu, plus 5 mM MgCl2 and 3 mM EGTA present, to eliminate calcium but keep divalent cations concentrated enough to avoid DNA dispersion.

In making up this solution:

  1. Use "EM grade" glutaraldehyde, usually supplied in concentrated form in sealed 2ml vials over argon or another inert gas.
  2. Be sure to add the MgCl2 and the EGTA before pH'ing the buffer to 7.4, or else the EGTA will not dissolve (or will drop the pH as it does dissolve). (We usually add the MgCl2 and the EGTA from unbuffered 1M stock solutions in distilled water.)
For fixing "whole", living cells, we suggest using:

2% glutaraldehyde in what we call "NaHCa" buffer. This consists of 30 mM HEPES buffer brought to pH 7.4 with NaOH (not KOH!), with 100 mM NaCl present to make it roughly isotonic and 2 mM CaCl2 present to preserve cell membranes.

In making up the buffer solution, to which the glutaraldehyde will be added later, be sure not to add the CaCl2 until after pH'ing the buffer to 7.4, or else the calcium will precipitate when the NaOH hits the surface of the solution. (We usually add the CaCl2 from an unbuffered 1M stock in distilled water.)

When adding the fixative itself to this buffer, use an "EM grade" of glutaraldehyde, usually supplied by commercial companies as a concentrated solution (25-70%) in a sealed 2ml or 10ml vial over argon or another inert gas.