Reference

Localization of human chorionic gonadotropin and placental lactogen by immunogold labeling for electron microscopy: technique and limitations

Localization of human chorionic gonadotropin and placental lactogen by immunogold labeling for electron microscopy: technique and limitations. Morrish, Donald W.; Marusyk, Halyna (Perinatal Res. Centre, Signal Transduction Lab., Univ. Alberta & W. W. Cross Cancer Inst., Edmonton, AB T6G 2S2, Can.). Microscopy Research and Technique, 38(1/2), 176-187 (English) 1997 Wiley-Liss. CODEN: MRTEEO. ISSN: 1059-910X. DOCUMENT TYPE: Journal; General Review CA Section: 2 (Mammalian Hormones) To demonstrate human chorionic gonadotropin (hCG) and human placental lactogen (hPL) secretory granules in placenta and to illustrate newer embedding techniques and specific immunospecificity problems in the placenta, labeling expts. using immunogold or peroxidase combined with avidin-biotin enhancement in epoxy LX-112-, Araldite-, or LR gold-embedded tissue fixed in 2.5% glutaraldehyde or 2.5% paraformaldehyde were carried out in term and first-trimester normal human placenta and in partial hydatidiform moles. Increased sensitivity of the low-temp. LR gold method was found for hPL-labeled granules. bHCG-labeled granules were noted in syncytium of first-trimester placenta, and bhCG-contg. granules in hydatidiform moles were similar to those of normal placenta. Paraformaldehyde fixation and LR gold embedding permitted identification of endoplasmic reticulum-assocd. labeling not obsd. with other methods. A brief review and discussion of immunolabeling methods, controls, and embedding materials is presented. The authors conclude that further refinement of peptide localization methods in the placenta is possible but must take into account the abundant potentially cross-reacting peptides present in the placenta.

Sequential paraformaldehyde and methanol fixation for simultaneous flow cytometric analysis of DNA, cell surface proteins, and intracellular proteins

Sequential paraformaldehyde and methanol fixation for simultaneous flow cytometric analysis of DNA, cell surface proteins, and intracellular proteins. Pollice, Agnese A.; McCoy, J. Philip, Jr.; Shackney, Stanley E.; Smith, Charles A.; Agarwal, Jyotsna; Burholt, Dennis R.; Janocko, Laura E.; Hornicek, Francis J.; Singh, Sarita G.; Hartsock, Robert J. (Allegheny-Singer Res. Inst., Allegheny Gen. Hosp., Pittsburgh, PA 15212, USA). Cytometry, 13(4), 432-44 (English) 1992. CODEN: CYTODQ. ISSN: 0196-4763. DOCUMENT TYPE: Journal CA Section: 9 (Biochemical Methods) A cell fixation and permeabilization procedure consisting of sequential paraformaldehyde and methanol was evaluated and found suitable for concomitant flow cytometric quantification of total cellular DNA, immunofluorescence measurements of cell surface proteins, and immunofluorescence measurements of intrazcellular proteins. Paraformaldehyde/methanol-fixed cells exhibited significantly greater intracellular antitubulin immunofluorescence than cell fixed with paraformaldehyde or methanol alone and significantly greater intracellular antitubulin immunofluorescence than cells fixed with methanol followed by paraformaldehyde. With paraformaldehyde/methanol fixation, cell morphol. was well preserved and forward and right angle light scatter properties were sufficiently well maintained to permit gating on these parameters. Cell surface marker staining with fluorescent anti-leukocyte antibodies was unaffected fixation with paraformaldehyde/methanol. Paraformaldehyde effects on the intensity of DNA staining with propidium iodide were dependent on paraformaldehyde concn. and fixation temp.; these effects were least pronounced at low paraformaldehyde concns. (0.25% or less), and at temps. >37°. Paraformaldehyde fixation may result in differences in propidium iodide staining of DNA in some diploid cells, which may produce small spurious aneuploid peakds in normal peripheral blood leukocytes. Paraformaldehyde fixation also produces an apparent increase in the DNA index of aneuploid cell populations in comparison with methanol fixation, particularly when the DNA index exceeds 1.5. Occasionally, this paraformaldehyde fixation-induced effects is useful in identifying biol. distinct near-diploid subpopulations in tumors.