Residual cyclic nucleotide associated with tissues after exposure to aqueous buffer analogous to that used in...

A question concerning cyclic nucleotide immunocytochemical localization has been how much nucleotide remains associated with the tissue section. To answer that question cryostat sections of goldfish eye and mouse spleen, liver and lung were mounted on microscope slides and air dried. Following fixation by a variety of procedures employing heat, paraformaldehyde, glutaraldehyde, acetone, or ethanol, the sections were exposed to buffer (20 microM NaHPO4, 154 microM NaCl, pH 6.1) for 4 hours. The tissues were then scraped from the slides into 0.4N Perchloric Acid and cAMP and cGMP extracted and measured. The results show that fractions of both nucleotides are retained during buffer exposure. However, the amount retained varied with the: i) neucleotide, ii) fixation procedure, and iii) tissue type. Cyclic GMP retention was consistently higher (20-70%) than cAMP (6-30%). Glutaraldehyde was consistently more efficient in fixing cAMP, while cGMP retention was more variable with different fixation procedures. Tissue variability is seen in the example that spleen and liver retained more cGmp (71.4 and 70.6% respectively) than lung and eye (22.8 and 37.7% respectively). Maximum nucleotide loss occured during the first 5-30 minutes of buffer exposure with no additional loss accuring after another 20 hours. Collectively, these results demonstrate that cyclic nucleotides are retained during immunocytochemical staining procedures but that the degree of retention is dependent on several variables.