Table 1.

Immunoreactive Peptide Levels in the Hypothalamus After Different Tissue Handling Procedures

Tissue handling procedure	DYNB	LARG	LARG/DYNB	MEAP
Nonstabilized brain
Standard procedure, freeze (n = 5)	20.1 ± 0.88	44.5 ± 1.83	2.23 ± 0.12	73.4 ± 2.18
Wet ice 15 minutes, freeze (n = 4)	18.8 ± 0.65	47.9 ± 2.50	2.55 ± 0.09	91.3 ± 7.06
Freeze, stabilize frozen before extraction (n = 6)	17.8 ± 1.48	7.29 ± 0.99**	0.37 ± 0.06**	221 ± 26.0**
Stabilize, freeze (n = 4)	35.1 ± 2.14*	2.28 ± 0.22*	0.07 ± 0.01*	418 ± 74.6*
Stabilized brain
Freeze (n = 4)	36.6 ± 1.05*	2.61 ± 0.18*	0.07 ± 0.01*	286 ± 47.8*
Wet ice 15 minutes, freeze (n = 4)	35.8 ± 4.79*	3.25 ± 0.11*,#	0.09 ± 0.01*	263 ± 15.9*

Stabilization was tested at different time points according to Figure 1 and the peptide levels were compared with standard procedure (in bold) that is, the brain was rapidly freed and the hypothalamus removed and rapidly frozen on aluminum-encased dry ice. In addition, the consequences of keeping the tissue samples on wet ice for 15 minutes were tested in nonstabilized and stabilized brain.

The values represent mean ± SEM and are expressed as fmol/mg tissue.

^*p < 0.05, ^**p < 0.01, compared to standard procedure; ^#p < 0.05, compared to immediately frozen hypothalamus from stabilized brain.

DYNB, dynorphin B; LARG, Leu-enkephalin-Arg⁶; MEAP, Met-enkephalin-Arg⁶-Phe⁷.