Addition of phosphotungstic acid to ethanol for dehydration improves both the ultrastructure and antigenicity of pituitary tissue embedded in LR White acrylic resin.
Although hydrophilic acrylic resins including LR White have been widely utilized as embedding media for immunocytochemical use, the constituents of tissues are often extracted by the resin monomer during the infiltration process of the embedment, resulting in a discernible impairment of the ultrastructure when the tissue is weakly fixed only with aldehydes. To minimize the extraction by the resin monomer, the embedding procedure with LR White resin was reexamined in the present study. Among the treatments tested, a partial dehydration with 70% ethanol containing 2% phosphotungstic acid (PTA) well preserved the ultrastructure of the pituitary tissue without spoiling the antigenicity of LHbeta and other representative markers for the Golgi apparatus. In addition, treatment with 1% tannic acid (TA) prior to the dehydration described above synergistically improved both the ultrastructure and antigenicity of the tissue so that the orientation of the Golgi apparatus could be determined by double immunogold labeling with commercially available anti-GM130 and anti-TGN38 antibodies. The ultrathin sections from the LR White-embedded tissue treated with TA and dehydrated in 70% ethanol containing 2% PTA also enhanced contrast without conventional heavy-metal staining with uranyl acetate and lead citrate. Our findings further suggest that the precipitation of TA and PTA protected the tissue from being extracted during the embedment, probably because an insoluble complex was transiently formed with the constituents of the tissue. This simple modification of the LR White embedment can extend the application of post-embedding immunocytochemistry as an alternative to pre-embedding immunolabeling with frozen ultrathin sections.
Sakai
Yuko
Y
Department of Anatomy II, Asahikawa Medical College, Midorigaoka-higashi 2-1-1-1, Asahikawa 078-8510, Japan.
Hosaka
Masahiro
M
Hira
Yoshiki
Y
Watanabe
Tsuyoshi
T
D000180:Acrylic Resins; D000818:Animals; D000941:Antigens / Q000276:immunology*; D003890:Desiccation; D000431:Ethanol / Q000737:chemistry*; D007150:Immunohistochemistry; D037101:Luteinizing Hormone, beta Subunit / Q000378:metabolism; D008297:Male; D010772:Phosphotungstic Acid / Q000737:chemistry*; D010902:Pituitary Gland / Q000276:immunology* / Q000648:ultrastructure*; D051381:Rats; D017208:Rats, Wistar; D016610:Tissue Embedding; D016707:Tissue Fixation
D000180:Acrylic Resins; D000941:Antigens; D037101:Luteinizing Hormone, beta Subunit; D010772:Phosphotungstic Acid; D000431:Ethanol; C048707:LR white