In situ hybridization for vasopressin mRNA in the human supraoptic and paraventricular nucleus; quantitative aspects of formalin-fixed paraffin-embedded tissue sections as compared to cryostat sections.
In order to study the suitability of formalin-fixed paraffin-embedded brain tissue for vasopressin (AVP)-mRNA detection, we used symmetric halves of 5 human hypothalami. In every case, one half was formalin fixed for 10-35 days and paraffin embedded while the other half was frozen rapidly. Following in situ hybridization (ISH) histochemistry on systematically obtained sections of the supraoptic (SON) and paraventricular nucleus (PVN) of both halves, total amounts of AVP-mRNA in these nuclei were estimated using densitometry of film autoradiographs. Total amounts of radioactivity were found to vary considerably between patients and amounted to 1297 +/- 302 arbitrary units (AU) (PVN) (mean +/- SEM) and 2539 +/- 346 (SON) for the cryostat sections and 868 +/- 94 (PVN) and 1259 +/- 126 (SON) for the paraffin tissue. Variations introduced by the method itself yielded a coefficient of variation of only 0.19. Furthermore, a non-significant negative trend with postmortem delay was found in cryostat tissue, but not in paraffin sections. No effect of fixation time was observed in the paraffin tissue. Both ways of tissue treatment have specific advantages and disadvantages that may be different for other probes or other brain areas. For ISH of a highly abundant mRNA like AVP in a very heterogeneous brain area such as the human hypothalamus, formalin-fixed paraffin-embedded tissue sections can be used for quantitative analysis of entire brain nuclei because of the small variation in this tissue, the remarkably good signal recovery (some 75% as compared to cryostat sections) and its practical advantages with regards to anatomical orientation, storage and sampling of the tissue.
Lucassen
P J
PJ
Graduate School Neurosciences Amsterdam, Netherlands Institute for Brain Research.
Goudsmit
E
E
Pool
C W
CW
Mengod
G
G
Palacios
J M
JM
Raadsheer
F C
FC
Guldenaar
S E
SE
Swaab
D F
DF
D005404:Fixatives; D005557:Formaldehyde; D006652:Histological Techniques; D006801:Humans; D017403:In Situ Hybridization; D016612:Paraffin Embedding; D010286:Paraventricular Hypothalamic Nucleus / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D013495:Supraoptic Nucleus / Q000378:metabolism*; D014667:Vasopressins / Q000235:genetics*
D005404:Fixatives; D012333:RNA, Messenger; D014667:Vasopressins; D005557:Formaldehyde