Immunoelectron Microscopy Study of Polyamines Using a Newly Prepared Monoclonal Antibody against Spermidine: Use of a Mixture of Glutaraldehyde and Paraformaldehyde as a Cross-Linking Agent in the Preparation of the Antigen

Abstract

We developed a mouse monoclonal antibody (ASPD-19, IgG3 sub-isotype mAb) against spermidine (Spd) conjugated to bovine serum albumin (BSA) using a mixture of glutaraldehyde (GA) and paraformaldehyde (PFA)-sodium borohydride for applications in immunoelectron microscopic studies. The antibody specificity was evaluated by an ELISA binding test simulating the immunocytochemistry (ICC) of tissue sections. The ASPD-19 mAb is highly specific for Spd and Spm, almost the same degree to each, and can distinguish alterations in the chemical structure of other polyamine (PA) analogs, showing less than 3.2% cross-reaction with N(1)-acetylspermidine, acetylspermine, or N(8)-acetylspermidine. By an indirect immunoperoxidase method using the ASPD-19 mAb, PA-like immunoreactivities were observed in different tissues fixed with Karnovsky fixative (a mixture of GA and PFA) in combination with borohydride reduction. In contrast, immunoreactivity was very low in tissues when the borohydride reduction step was omitted. The PA-like immunoreaction was completely abolished by the adsorption of the ASPD-19 mAb with 100 microg/ml of Spd or Spm, but was inhibited little or none by other PA-related compounds or amino acids. A light microscopic ICC method using ASPD-19 produced immunostaining of PAs in certain cells in rat tissues with high biosynthetic activities (small intestine, pancreas and spinal cord). A pre-embedding immunoelectron microscopic study using rat spinal cord showed PA immunoreactivity located predominantly on free (polysomes) and attached ribosomes of the rough endoplasmic reticulum (Nissl bodies) in the cytoplasm of motor neurons. These results are in complete agreement with the results obtained by our recent ICC method using another mAb (ASPM-29) produced against GA-conjugated Spm.

Polyamines in spermatocytes and residual bodies of rat testis

“…In thusdeWned residual bodies, RNA colocalized together with PAs and ultrastructural immunocytochemistry demonstrated that such colocalization reXected an association between PAs and ribosomes. This is in good agreement with our previous results showing PA localization on ribosomes in neurons and gastrointestinal epithelial cells (Fujiwara et al 1998;Tanabe et al 2004;Shin et al 2006). Since residual bodies are formed from maturing spermatids it is a triXe surprising that the latter cells stained weaklier for PAs.…”

“…Immunocytochemical localization of PAs may help to unravel their true biological roles by revealing their exact cellular and subcellular localization. We have previously demonstrated that PAs are localized in association with ribosomes in neurons and gastrointestinal epithelial cells (Fujiwara and Masuyama 1995;Fujiwara et al 1998;Tanabe et al 2004;Shin et al 2006): localization in agreement with biochemical studies suggesting a role for PAs in protein synthesis Tabor 1984, 1985;Pegg 1986;Yoshida et al 2004).…”

Immunoelectron microscopic study of polyamines in the gastrointestinal tract of rat

“…Immunocytochemistry (ICC) studies of PAs will help in developing a better understanding of these functional roles by revealing the exact cellular and subcellular localization of endogenous PAs. A PA ICC method had been previously developed, originally using polyclonal anti-Spm serum (Fujiwara et al 1983;Hougaard et al 1986Hougaard et al , 1987 and, only recently, using monoclonal antibodies (mAbs) ASPM-29, ASPD-19, and APUT-32, produced against conjugated Spm, Spd, and Put via glutaraldehyde (GA), a mixture of GA and paraformaldehyde (PFA) (Karnovsky fixative), and GA, respectively (Fujiwara and Masuyama 1995;Fujiwara et al 2001, Tanabe et al 2004). The former two mAbs specific for Spm and Spd, and the latter specific for Put, have proven to be extremely useful for their localization in a variety of cells and tissues in ICC studies.…”

Immunocytochemical demonstration of polyamines in nucleoli and nuclei

“…Using the same principle, we have previously demonstrated that PAs occur associated with both free and ER-bound ribosomes in the cytoplasm of any cell type (Fujiwara and Masuyama 1995;Fujiwara et al 1998a;Tanabe et al 2004;Shin et al 2006Shin et al , 2007. Hougaard et al (1987) have reported on nuclear localization of PAs in chick erythrocytes, and more recently, Johnson et al (2004) reported on nuclear staining for PAs in cell lines, although their data were not supported by speciWcity (absorption) controls.…”

“…This ICC system has proved useful for detecting PAs in cells and tissues (Fujiwara et al 1996(Fujiwara et al , 1997a. Moreover, by immunoelectron microscopy, this ICC system revealed that PAs are associated with both free and ER-bound ribosomes in all cell types investigated (Fujiwara et al 1998a;Tanabe et al 2004;Shin et al 2006Shin et al , 2007. Since ribosomes originate from nucleoli, we asked ourselves whether PAs also were associated with nucleoli and other regions of the nucleus.…”