Detection of oxytocin mRNA in hypertonic saline Fos-activated PVN neurons: comparison of chromogens in dual immunocytochemical and in situ hybridization procedure.
OBJECTIVE
The aim of the present experiments was to elucidate: 1. the stability and usefulness of a 3,3 -diaminobenzidine tetrahydrochloride (DAB) chromogen intensified with nickel and cobalt (DAB-Ni-Co) in the dual immunocytochemical and in situ hybridization procedure using Fos-protein antibody and oxytocin mRNA (OXY mRNA) radiolabeled probe; 2. the susceptibility of the free floating and mounted cryostat sections, freshly prepared or stored for 24 month at -20 degrees C.


METHODS
The dual staining procedure was tested on neurons of the hypothalamic paraventricular nucleus (PVN) activated by an intraperitoneal injection of hypertonic saline (HS, 1.5 M, 5 ml, 60 min). Two dual labeling procedures were compared: 1/ Fos-immunostaining with DAB alone and combined with OXY mRNA in situ hybridization and 2/ Fos-immunostaining with DAB-Ni-Co and combined with OXY mRNA in situ hybridization. In both experiments free floating and mounted cryostat sections, freshly prepared or stored for 24 month at -20 degrees C, were tested.


RESULTS
HS strongly stimulated both the parvicellular and magnocellular population of PVN neurons followed by an extensive Fos-immunolabeling in many cell nuclei. The first staining sequence with Fos-DAB labeling resulted in a good staining quality on both the fresh and for 24 month stored mounted sections. Although the free floating sections during the in situ procedure showed the same staining properties as the mounted ones, with respect to their increased fragility on the end of the hybridization procedure, they were difficult to mount and stretch on poly-L-lysine coated slides. On the other hand, Fos-immunolabeling with DAB-Ni-Co exhibited improved staining density of the single DAB chromogen in the first staining sequence of the dual staining procedure. However, DAB-Ni-Co mixture showed up as an unstable chromogen complex, which after completing the in situ hybridization process completely disappeared from each type of section.


CONCLUSIONS
The results of the present dual immunocytochemical-in situ hybridization staining utilizing Fos-antibody and OXY mRNA oligoprobe indicate that this procedure is applicable on free floating as well as mounted cryostat sections, freshly prepared or stored for 24 month at -20 degrees C. However, the dual procedure is only successful when the immunoproduct in the first sequence is visualized with an unintensified DAB and not with combined DAB-Ni-Co chromogen and when the histological sections in the second sequence are not processed as free floating but are attached to a poly-L-lysine coated microscopic glasses.
Pirnik
Zdeno
Z
Institute of Experimental Endocrinology, Slovak Academy of Sciences, Vl rska 3, 833 06 Bratislava, Slovak Republic. ueenpirn@savba.sk
Kiss
Alexander
A
D015100:3,3'-Diaminobenzidine; D000818:Animals; D001345:Autoradiography; D002863:Chromogenic Compounds; D003035:Cobalt; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D008297:Male; D009474:Neurons / Q000737:chemistry* / Q000187:drug effects; D009532:Nickel; D010121:Oxytocin / Q000235:genetics*; D010286:Paraventricular Hypothalamic Nucleus / Q000166:cytology; D016760:Proto-Oncogene Proteins c-fos / Q000032:analysis*; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D017208:Rats, Wistar; D012462:Saline Solution, Hypertonic
D002863:Chromogenic Compounds; D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger; D012462:Saline Solution, Hypertonic; D015100:3,3'-Diaminobenzidine; D003035:Cobalt; D010121:Oxytocin; D009532:Nickel