Microwave pretreatment of sections to improve the immunocytochemical detection of progesterone receptors in the guinea pig hypothalamus.
The goal of this study was to evaluate the effects of microwave heat on immunocytochemical staining for progesterone receptors (PR) in frozen sections and in paraffin-embedded sections of guinea pig brain using an antibody previously shown to successfully stain only frozen sections of formalin-fixed materials. A simple, reliable and sensitive retrieval method has been adapted based on microwave irradiation of sections in citrate solution prior to incubation with the anti-PR antibody. Different pH values (3.0, 6.0 and 8.5) of the citrate buffer were tested and the pH at 6.0 was found to give the best results. PR-like immunoreactivity was markedly increased in frozen sections exposed to microwave heating compared with untreated tissue. The integrity of the tissue was not affected by this treatment and specific nuclear immunoreactivity for PR was detected in hypothalamic neurons. Staining of paraffin-embedded sections without microwave pretreatment yielded negative results while irradiation of sections enabled the labeling of hypothalamic regions known to contain progesterone binding sites. Immunocytochemical staining did not reveal any significant difference in the number of PR-immunoreactive neurons between frozen tissue compared with paraffin-embedded tissue. The results indicate that microwave irradiation can unveil PR antigenicity in sections of paraffin-embedded brain and enhances immunostaining in sections of frozen tissue.
Warembourg
M
M
INSERM U422, 1 Place de Verdun, 59045 Cedex, Lille, France. warembourg@lille.inserm.fr
Leroy
D
D
D000818:Animals; D002021:Buffers; D002951:Citrates / Q000032:analysis; D005260:Female; D006168:Guinea Pigs; D006863:Hydrogen-Ion Concentration; D007031:Hypothalamus / Q000166:cytology / Q000378:metabolism*; D007150:Immunohistochemistry; D008867:Microtomy / Q000379:methods*; D008872:Microwaves; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D011980:Receptors, Progesterone / Q000032:analysis*; D000077559:Sodium Citrate
D002021:Buffers; D002951:Citrates; D011980:Receptors, Progesterone; D000077559:Sodium Citrate