Immunofluorescence technique for 100-nm-thick semithin sections of Epon-embedded tissues.
An immunofluorescence staining method for Epon-embedded materials is described. Rat kidney and liver were fixed by perfusion with 1% glutaraldehyde for 10 min. Tissue slices were embedded in Epon. Semithin sections with thicknesses ranging from 1,000 to 100 nm were cut and mounted on clean glass slides. Epoxy resin was removed by treatment with 10% sodium ethoxide. Sections were digested with 0.05% trypsin and then treated with sodium borohydride. Sections were immunostained for leucine aminopeptidase (plasma membrane), catalase (peroxisomes), 3-ketoacyl-CoA thiolase (mitochondria), cathepsin D (lysosomes), and LGP107 (lysosomal membrane) using Cy(2)- or Alexa 546-labeled secondary antibodies. In 1,000-nm-thick sections, non-specific fluorescence remained and such fluorescence decreased as the sections became thinner. Clear specific fluorescence was obtained in the sections with thicknesses ranging from 250 to 100 nm with Alexa 546-labeled antibody (red fluorescence) but was not specific enough with Cy(2)- or Alexa 430-labeled antibody (green fluorescence). Sodium borohydride greatly abolished autofluorescence of glutaraldehyde. The present method made it possible to obtain signals in cross-sections of biological materials with a thickness of 250-100 nm, which are difficult to obtain in optical section using a confocal laser microscope.
Haraguchi
Celina M
CM
Biology Laboratory, Yamanashi Medical University, Tamaho-cho, Yamanashi 409-3898, Japan.
Yokota
Sadaki
S
D000102:Acetyl-CoA C-Acyltransferase / Q000032:analysis; D000818:Animals; D002374:Catalase / Q000032:analysis; D002402:Cathepsin D / Q000032:analysis; D004853:Epoxy Resins; D005455:Fluorescent Antibody Technique / Q000379:methods*; D007668:Kidney / Q000737:chemistry*; D007931:Leucyl Aminopeptidase / Q000032:analysis; D008099:Liver / Q000737:chemistry*; D008247:Lysosomes / Q000737:chemistry; D008856:Microscopy, Fluorescence; D008867:Microtomy; D051381:Rats; D016610:Tissue Embedding / Q000379:methods
D004853:Epoxy Resins; C004875:EPON; D002374:Catalase; D000102:Acetyl-CoA C-Acyltransferase; D007931:Leucyl Aminopeptidase; D002402:Cathepsin D