Immunogold labeling

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 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. 

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. 

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. 

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. 

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. 

Vandenbosch KA. Immunogold labeling, in Electron Microscopy of Plant Cells (Hall JL, Hawes C, eds.), Academic Press, San Diego, CA, 1991, pp. 181-218. 

Fig. 12.2 Immunogold localization of endothelial NO synthase (NOS3) in skeletal muscles, a Immunogold labeling of NOS3 in subsarcolemmal mitochondria, b Inside of the sarcoplasm NOS3 is localized along the contractile fibers, in the sarcoplasmic reticulum and in mitochondria. EPON embedding, etching with sodium ethoxide, antigen retrieval in citrate buffer, pH 6.0. 12 nm immunogold.

Place the microtubes with grids at room temperature and allow them to cool for about 15 20 min. Before the beginning of the immunogold labeling protocol (see below), grids should be washed by dipping them repeatedly in water for 45 60 s. 

ICC of astrocyte cell culture shows CYP2E1 over cytoplasm and processes but more pronounced over nuclear membrane immunogold labeling agrees. Ethanol causes an increase in CYP2E1 as determined by ICC and Dot blot (Montoliu et al., 1995). 

Birrell, G. B., Hedberg, K. K., and Griffith, P. H. (1987) Pitfalls of immunogold labeling analysis by light microscopy, transmission electron microscopy, and photoelectron microscopy. J. Histochem. Cytochem. 35, 843-853. 

Figure 6. A Photomicrograph (x 51,000) of caffeine treated leaf epidermal cell showing electron-dense deposits on cell wall and membrane vesicles fusing with the plasmalemma (arrows). B Immunofluorescence labeling of flavonoids in cell walls of leaf epidermal strips (arrows) and autofluorescent stomata (x 62.5). C Immunogold labeling of the walls of a mesophyll cell (left, x 41,000). Ch, chloroplast EC, epidermal cell G, Golgi IS, intercellular space MC, mesophyll cell (right, control x 19,500).

Post-staining Procedure. Immunogold labelled sections were post-stained in saturated aqueous uranyl acetate for 15 min and lead citrate for 10 min... 

Transmission electron microscopy of immunogold labelled sections has shown that the extracellular lignin-degrading enzymes lignin-peroxidase and laccase were localized within the cell wall and mucilage of the hyphae of C. versicolor. Laccase was present in the cell wall layer whereas lignin-... 

Rash JE, Yasumura T, Hudson CS, Agre P, Nielsen S (1998) Direct immunogold labeling of aquaporin-4 in square arrays of astrocyte and ependymocyte plasma membranes in rat brain and spinal cord. Proc Natl Acad Sci USA 95 11981-11986... 

Unlike Euglena PNO, which is a mitochondrial protein (Rotte et al. 2001), CpPNO lacks a mitochondrial targeting presequence and does not localize within the relic organelle (Ctrnacta et al. 2006). In fact, sporozoites of C. parvum visualized both by confocal immunofluorescence (Fig. 3) and immunogold-labelled (Fig. 4) confirm that CpPNO has an unique com-partmentalization firstly within the cytosol as expected, but secondly within... 

Fig. 5.3 Immunogold labeling of human A i ARs on ultrathin sections of transfected CHO cells, (a) Small aggregates of gold particles (arrow) on the plasma membrane after a 60-min incubation with 10 nM agonist NECA at 4°C. (b) After a 15-min incubation with agonist at 37°C A3 ARs are visible in uncoated vesicle at level of the cortical cytoplasm (Trincavelli et al. 2000)...

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