Immunogold

Further studies have shown that D. africanus ferredoxins I and II are involved as physiological electron carriers of POR. Also, using immunogold labeling, it was possible to show that POR is located in the cytoplasm. 

By combining our images of these walls and their constituent polymers during chemical extraction, with data from immunogold labelling of thin-sectioned material, we were able to construct a simple structural model of the primary cell wall of onion (Fig 3). 

Fig 2 Immunogold negative staining, with a monoclonal antibody (JIM 5 (13)) that recognises a relatively unesterified pectic epitope, of rhamnogalacturonans extracted from onion cell walls. Arrows indicate 5 nm colloidal gold particles. Scale bar represents 200nm. 

Fig 5 Immunogold labelling on thin sections of low-temperature embedded cell walls from 9-day old tobacco cells with JIM 5, a monoclonal antibody that recognises a relatively unesterified pectic epitope. Cell walls of elongating cells label very weakly, but material that is being secreted into the culture medium labels strongly. The old part of the wall is labelled but new wall material is not. 

Immunogold localization of the pectic epitope has been performed on different types of cells cell suspensions, roots, shoots, meristems, coleoptiles, pollen grains, protoplasts from different species carrot, sugar beet, tobacco, oat... The pattern of labeling was always the same polygalacturonic acid was essentially located on the material expanded at three-way junctions between cells or lining intercellular space, but was not found in primary walls. No epitope could be located close to the plasma membrane (Fig. lO.a). Middle lamellae far from junction zones and walls of meristematic cells were never labeled. 

Fig. 10. Intercellular junction zones of carrot cells grown in suspension have been observed in electron microscopy after immunogold labeling with the 2F4 antibody, (a) no treatment of the sections prior to labeling the gold particles are restricted to the center of the junction zones (b) enzymatic (pectin methyl esterase) deesterification of the E.M. grids before labeling the deesterified pectins present in the primary walls now bind the probe. Scale bars = 1 pm.

Immunogold labeling with JIM S exhibited an identical labeling distribution for polygalacturonic acid as was obtained indirectly with the EPG EMSIL (inset of Fig. 1). Control experiments for labeling specificities obtained by the direct or indirect methods resulted in total elimination of specific labeling. The cellulase-gold probe heavily labeled the epidermal cell walls (Fig. 2). 

NPQ (Rakhimberdieva et al. 2004) exactly matches the absorption spectrum of the carotenoid, 3 -hydrox yech i nenone (Polivka et al. 2005) in the OCP. The OCP is now known to be specifically involved in the phycobilisome-associated NPQ and not in other mechanisms affecting the levels of fluorescence such as state transitions or D1 damage (Wilson et al. 2006). Studies by immunogold labeling and electron microscopy showed that most of the OCP is present in the interthylakoid cytoplasmic region, on the phycobilisome side of the membrane, Figure 1.2 (Wilson et al. 2006). The existence of an interaction between the OCP and the phycobilisomes and thylakoids was supported by the co-isolation of the OCP with the phycobilisome-associated membrane fraction (Wilson et al. 2006, 2007). 

FIGURE 1.2 In situ localization of the OCP-green fluorescence protein (GFP) fusion protein Immunogold labeling of a thin section of OCP-GFP transformed Synechocystis PCC6803 OCP-GFP cells were labeled with a polyclonal antibody against the GFP coupled to 10 nm gold particles. Bar = 0.5 pm. 

Fig. 10.2. Immunogold staining of an ultra-thin section of an immature Acanthocheilonema viteae uterine mf with mAb 24-4. Note that A. viteae chitinase is present in the cuticle (arrowheads), but not on the surface. (Photograph W. Rudin.)...

Ramandeep, Dikshit KL, Raje M. Optimization of immunogold labeling TEM an ELISA-based method for rapid and convenient simulation of processing conditions for quantitative detection of antigen. J. Histochem. Cytochem. 2001 49 355-367. 

Goode NP, Shires M, Crellin DM, et al. Post-embedding double-labeling of antigen-retrieved ultrathin sections using a silver enhancement-controlled sequential immunogold (SECSI) technique. J. Histochem. Cytochem. 2004 52 141-144. 

Ni J., Lipert R.J., Dawson G.B., Porter M.D., Immunoassay readout method using extrinsic Raman labels adsorbed on immunogold colloids, Anal. Chem. 1999 71 4903-4908. 

Cramer, E.M., Beesley, J.E., Pulford, K.A.F., Breton-Gorius, J., and Mason, D.Y. (1989) Colocalization of elastase and myeloperoxidase in human blood and bone marrow neutrophils using a monoclonal antibody and immunogold. Am. J. Pathol. 134, 1275-1284. 

Cubie, H.A., and Norval, M. (1989) Detection of human papilloma viruses in paraffin wax sections with biotinylated synthetic oligonucleotide probes and immunogold staining./. Clin. Pathol. 42, 988-991. 

De Waele, M., Renmans, W., Segers, E., De Valck, V., Jochmans, K., and van Camp, B. (1989) An immunogold-silver staining method for detection of cell surface antigens in cell smears. J. Histochem. Cytochem. 37, 1855-1862. 

Ellis, I.O., Bell, J., and Bancroft, J.D. (1988) An investigation of optimal gold particle size for immuno-histological immunogold and immunogold-silver staining to be viewed by polarized incident light (EPI polarization) microscopy./. Histochem. Cytochem. 36, 121-124. 

Gillitzer, R., Berger, R., and Moll, H. (1990) A reliable method for simultaneous demonstration of two antigens using a novel combination of immunogold-silver staining and immunoenzymatic labeling. J. Histochem. Cytochem. 38, 307-313. 

Holgate, C., Jackson, P., Cowen, P., and Bird, C. (1983) Immunogold-silver staining new method of immunostaining with enhanced sensitivity./. Histochem. Cytochem. 31, 938. 

Martinez-Ramon, A., Knecht, E., Rubio, V., and Grisolia, S. (1990) Levels of carbamoyl phosphate synthetase I in livers of young and old rats assessed by activity and immunoassays and by electron microscopic immunogold procedures. J. Histochem. Cytochem. 38, 371-376. 

Moeremans, M., Daneels, G., Van Dijck, A., Langanger, G., and De Mey, J. (1984) Sensitive visualization of antigen-antibody reactions in dot and blot immuno overlay assays with the immunogold and immu-nogold/silver staining./. Immunol. Meth. 74, 353-360. 

Nielsen, M.H., Bastholm, L., Chatterjee, S., Koga, J., and Norrild, B. (1989) Simultaneous triple-immunogold staining of virus and host cell antigens with monoclonal antibodies of virus and host cell antigens in ultrathin cryosections. Elistochemistry 92, 89-93. 

Fig. 8.2 HSA accumulation in transgenic chloroplasts. (A-C) Electron micrographs of immunogold-labeled tissues from untransformed leaves (A) and mature leaves transformed with the chloroplast vector pLDApsbAHSA (B-C). Magnifications A x 10000 B x 5000 C x 6300.

In addition to fluorescence methods, another study [27] developed a method to permit electron microscopic localization of Ras anchor domains on cytoplasmic membrane surfaces by immunogold labeling. The particle neighbor distances can be analyzed to obtain information about possible domain structure. Expressing H-Ras and K-Ras in baby hamster kidney cells, a nonrandom particle distribution was obtained from which the estimated mean raft size was 7.5-22 nm and about 35% of the membrane area consists of rafts. The same technique applied to cells that had been incubated with [3-cydodextrin to reduce cholesterol produced completely random distributions of H-Ras. This cholesterol dependence suggests some type of coupling of rafts across the inner and outer membrane leaflets. 

Tao-Cheng, J. H. and Zhou, F. C. Differential Polarization of serotonin transporters in axons versus soma-dendrites an immunogold electron microscopy study. Neuroscience 94 821-830, 1999. 

Leino,R.L.,Gerhart,D.Z.andDrewes,L.R.Monocarboxylate transporter (MCT1) abundance in brains of suckling and adult rats a quantitative electron microscopic immunogold study. Brain Res. Dev. Brain Res. 113 47-54,1999. 

Bergersen, L., Rafiki, A. and Ottersen, O. P. Immunogold cytochemistry identifies specialized membrane domains for monocarboxylate transport in the central nervous system. Neurochem. Res. 27 89-96, 2002. 

Nixon GF, Mignery GA, Somlyo AV 1994 Immunogold localization of inositol 1,4,5-trisphosphate receptors and characterization of ultrastructural features of the sarcoplasmic reticulum in phasic and tonic smooth muscle. J Muscle Res Cell Motil 15 682-700 Peng H, Matchkov V, Ivarsen A, Aalkjaer C, Nilsson H 2001 Hypothesis for the initiation of vasomotion. CircRes 88 810-815... 

Nixon We saw the same with immunogold labelling, but looking with confocal microscopy we saw some InsP3 receptors around the nuclear envelope they look to be closely associated with it. When we do osmium ferrocyanide staining of the SR, the nuclear envelope does stain the same as the SR, so you would predict that there probably would be some there. 

Nixon The problem is that we lack sufficient resolution. Immunogold labelling should give the required resolution, but technically we have been unable to find evidence for them being closely localized. 

Hyatt AD. Immunogold labeling techniques, in Electron Microscopy in Biology— A Practical Approach (Harris JR, ed.), IRL Press, Oxford, UK, 1991, pp. 59-81. 

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