AN ARTEFACT IN RADIOAUTOGRAPHY DUE TO BINDING OF FREE AMINO ACIDS TO TISSUES BY FIXATIVES

TLDR

High free substrate levels during fixation can cause artifact binding that must be controlled in radioautography. The study examined how common fixatives bind labeled free amino acids to liver tissue and purified protein. Liver slices were incubated for 2 min with labeled leucine and puromycin, allowing leucine uptake but blocking protein incorporation, to assess fixative binding. Glutaraldehyde binds far more free leucine than formaldehyde (≈25 % vs 0.5 %) and produces 30‑fold more free‑amino‑acid binding in radioautographs (63 % of grains) compared to formaldehyde (4 %), while osmic acid shows intermediate binding; thus formaldehyde is the preferred fixative for radioautography.

Abstract

The binding of labeled free amino acids to liver and to purified protein by commonly used fixatives was investigated. Glutaraldehyde caused 25% of free leucine to be bound to serum albumin in solution, whereas formaldehyde bound only 0.5%. Liver slices were incubated for 2 min in the presence of labeled leucine and of puromycin, which permits absorption of leucine into the cell but inhibits incorporation into protein. Both counting and radioautographic techniques showed that glutaraldehyde bound 30 times, and osmic acid six times, as much free amino acid as did formaldehyde. By comparing liver slices incubated with and without puromycin for 2 min, it was calculated that in radioautographs prepared after fixation with glutaraldehyde, osmic acid, or formaldehyde 63, 25, and 4% respectively of the grains were due to binding of free amino acid. Formaldehyde, freshly prepared from paraformaldehyde, gives good preservation and is the recommended fixative for radioautography. When levels of free substrate in a tissue are high at the time fixative is added, the amount of binding of free substrate induced by the fixative should be included as a control in radioautographic experiments.