Antisera to Glutathione: Characterization and Immunocytochemical Application to the Rat Cerebellum

Abstract

Rabbits were immunized with reduced glutathione (gamma-glutamyl-cysteinyl-glycine) coupled to bovine serum albumin by glutaraldehyde or a mixture of glutaraldehyde and formaldehyde. The antisera that were formed were tested qualitatively, by screening them against more than 50 amino acids and peptide conjugates that had been immobilized on cellulose discs (spot test), and quantitatively, by immunogold analysis of test conjugates that had been embedded in an epoxy resin. It was shown that the antisera selectively recognized the reduced and oxidized forms of glutathione and that they did not exhibit any significant crossreactivity with glutamate, cysteine, glycine, gamma-glutamyl-cysteine or cysteinyl-glycine. Immunocytochemistry of Vibratome sections of rat cerebellum suggested that glutathione occurs in glial cells as well as in neurons. This was confirmed by electron microscopic, immunogold cytochemistry of tissue from rat cerebellum that had been freeze-substituted and embedded in Lowicryl under low temperature. Gold particles were concentrated over Golgi epithelial cells and perivascular glial processes, but also occurred over several types of neuronal profile including Purkinje and granule cell bodies, and mossy fibre terminals. At the subcellular level, glutathione-like immunoreactivity was found in the cytoplasmic matrix, mitochondria and nuclei. The immunolabelling intensity was strongly reduced in animals that had been pretreated with buthionine sulphoximine, which is known to depress the level of glutathione by inhibiting gamma-glutamyl-cysteine synthetase. The availability of antisera to glutathione is likely to further our understanding of the physiological and pathophysiological roles of this tripeptide.

Organization of Ionotropic Glutamate Receptors at Dendrodendritic Synapses in the Rat Olfactory Bulb

“…Specimens from the main olfactory bulb were rinsed in PB with 4% glucose (4°C, overnight), cryoprotected in increasing concentrations of glycerol (10,20,and 30% in PB), and rapidly frozen in liquid propane in a cryofixation unit (K F80; Reichert, Vienna, Austria). They were then freeze-substituted with methanol and embedded in L owicryl HM20 (L owi, Waldkraiburg, Germany), as described previously (Hjelle et al, 1994;Chaudhry et al, 1995).…”

Loss of perivascular aquaporin 4 may underlie deficient water and K + homeostasis in the human epileptogenic hippocampus

“…Small tissue blocks (0.3 ϫ 0.5 ϫ 1 mm 3 ) of area CA1 were dissected from the 500-m Vibratome sections and subjected to freeze substitution (15). Briefly, the tissue blocks were cryoprotected in glycerol and rapidly frozen in liquid propane at Ϫ190°C.…”

Carbonic anhydrase XIV is enriched in specific membrane domains of retinal pigment epithelium, Müller cells, and astrocytes

“…For electron microscopic immunocytochemistry, small blocks of the eyecup were subjected to freeze substitution and infiltrated in Lowicryl as described (23). Ultrathin sections were processed for immunogold cytochemistry (24) with the CA XIV antibody (1:200) followed by gold-conjugated secondary antibody (15-nm particles).…”