The extraction and assay of substance P in the rat sciatic nerve.
Published data for substance P (SP) content of sciatic nerves in control rats vary greatly. This study sought possible reasons for this variability by examining the influence of homogenisation procedures, freezing and selection of left/right nerve or proximal/distal segments. Substance P-like immunoreactivity (SP-LI) content (pg/mg nerve protein +/- SD) was significantly greater in sciatic nerves which had been homogenised using motor-powered equipment (615.4 +/- 146.3) as opposed to a hand-held pestle (445.4 +/- 111.8). Our second investigation revealed that freshly homogenised nerve tissue yielded greater SP-LI (508.8 +/- 88.7) than either tissue snap frozen in liquid nitrogen (307.6 +/- 77.9), snap-frozen and stored at -70 degrees C for 7 days (331.8 +/- 53.5), or tissue allowed to remain in the cadaver for 1 h and subsequently dissected and homogenised immediately (412.6 +/- 105.8). These data also show that storage at -70 degrees C imposes no further losses on those caused by freezing and extraction of frozen tissue. Two further studies indicated no variation between left and right sciatic nerves nor any proximal-distal gradients. Hence, this study illuminated the need for samples to be homogenised using motor-powered equipment immediately upon dissection, followed by SP extraction, for complete avoidance of losses of SP-LI.
Singh
J K
JK
Department of Pharmacology, Faculty of Basic Medical Sciences, Queen Mary and Westfield College, London, UK.
Diemel
L T
LT
Willars
G B
GB
Tomlinson
D R
DR
D000818:Animals; D005615:Freezing; D008297:Male; D011817:Rabbits; D011863:Radioimmunoassay; D051381:Rats; D017208:Rats, Wistar; D012015:Reference Standards; D015203:Reproducibility of Results; D012584:Sciatic Nerve / Q000737:chemistry*; D013373:Substance P / Q000032:analysis* / Q000302:isolation & purification; D014020:Tissue Extracts
D014020:Tissue Extracts; D013373:Substance P