N A N O P R O B E S     E - N E W S

Vol. 7, No. 12          December 21, 2006

Noxious stimuli are sensed and carried to the spinal cord dorsal horn by Adelta and C primary afferent fibers. Some of this input is relayed directly to supraspinal sites by projection neurons, while much of it impinges on a heterogeneous population of interneurons in lamina II. G-protein-gated inwardly rectifying potassium (GIRK) channels have been shown to be expressed in lamina II of the mouse spinal cord, and pharmacologic ablation of spinal GIRK channels has been found to selectively blunt the analgesic effect of high - but not lower - doses of intrathecal µ-opioid receptor (MOR) agonists.

GIRK channels formed by GIRK1 and GIRK2 subunits have now been found in two large populations of lamina II excitatory interneurons. Electrophysiological studies showed that one population displays relatively large apparent whole-cell capacitances and prominent GIRK-dependent current responses to the MOR agonist [D-Ala2,N-MePhe4,Gly-ol 5]-enkephalin DAMGO). A second population shows smaller apparent capacitance values, and exhibits a GIRK-dependent response to the GABAB receptor agonist baclofen, but not to DAMGO.

Immunoelectron microscopy with Nanogold® labeling was used to localize the GIRK subunits and determine their distribution in these two populations, via ultrastructural analysis using a pre-embedding approach. Free-floating sections were incubated in 10% normal goat serum (NGS) diluted in Tris-buffered saline (TBS) for 1 hour at room temperature, then incubated for 48 hours at 4°C with primary antibodies (GIRK1 or GIRK2) at final concentrations of 12 µg/ml diluted in TBS/1% NGS. GIRK subunit immunoreactivity was revealed by silver-intensified immunogold reaction: sections were incubated for 3 hours at room temperature with goat Nanogold-Fab' anti-rabbit or biotinylated goat anti-rabbit Fab fragments. All secondary antibodies were diluted 1:100 in TBS/1% NGS. Sections were then washed with TBS and double-distilled water, followed by silver enhancement of the gold particles with HQ Silver for 810 min. After washing in PBS and postfixing with OsO₄ (1% in 0.1 M phosphate buffer) and block staining with uranyl acetate, the sections were dehydrated in graded series of ethanol solutions, and flat-embedded on glass slides in Durcupan resin. Regions of interest were cut at 7090 nm with an ultramicrotome. Ultrathin sections were mounted on 200-mesh nickel grids, and counterstained on drops of 1% aqueous uranyl acetate, followed by Reynoldss lead citrate. Electron microscopic samples were obtained from three different mouse spinal cords, and three blocks of each animal were cut for electron microscopy.

Ultrastructural analysis was performed with a TEM-JEOL 100-CX electron microscope (JEOL, Peabody, MA), and photomicrographs captured with a CCD camera and digitally modified for brightness and contrast. To assess the specificity of GIRK subunit antisera, corresponding sections from GIRK knock-out mice were processed in parallel. Labeled structures were classified as axons, axon terminals, synapses, dendrites, or astrocytic processes based on morphological information in each section: axons were identified by their lack of synaptic input, the presence of neurotubules and occasional vesicles, and, in rare instances, presence of myelin. Axon terminals were identified by the presence of synapses and small round and/or large granular vesicles and synapses identified as parallel membranes separated by widened clefts associated with membrane specializations. Synapses displaying a prominent density on the postsynaptic side were characterized as asymmetrical (putative excitatory), while those showing equivalent densities on both sides were characterized as symmetrical (putative inhibitory). Smaller axon terminals that contained flattened vesicles and establish a single synapse were also identified in lamina II. These structures are thought to represent the terminal endings of intrinsic GABAergic interneurons. Dendrites were identified by the presence of synaptic contacts, lack of small vesicles, diffuse filaments, and numerous mitochondria. Astrocytic processes were identified by their amorphous shape, lack of vesicles and synaptic contacts, and presence of glial microfilaments.

Ultrastructural analysis revealed that GIRK subunits preferentially label type I synaptic glomeruli. This suggests that GIRK-containing lamina II interneurons receive prominent input from C fibers, and little input from Adelta fibers. Thus, excitatory interneurons in lamina II of the mouse spinal cord can be subdivided into different populations based on the neurotransmitter system coupled to GIRK channels. This important distinction provides a unique opportunity to characterize and relate spinal nociceptive circuitry according to a defined physiological significance.

Reference:

• Marker, C. L.; Lujan, R.; Colon, J., and Wickman, K.: Distinct populations of spinal cord lamina II interneurons expressing G-protein-gated potassium channels. J. Neurosci., 26, 12251-12259 (2006).
  • Abstract (courtesy of the Journal of Neuroscience).

More information:

• Nanogold Conjugates: catalog information
• Nanogold-antibody conjugates: product information and instructions
• Pre-embedding Nanogold immunolabeling with HQ Silver enhancement: special feature
• HQ Silver: product information and instructions
• Technical help - Nanogold conjugates
• Introducing Nanogold-Fab': our paper from Microscopy and Microanalysis 1991
• Nanogold labeling references, sorted by application

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Troubleshooting: Low or No Labeling with Nanogold® Conjugates

There are several reasons for low labeling, or an apparent failure to label, and the failure may be due to one of several components. Working through the following checklist will help you to identify the cause of your low labeling in the most efficient manner, losing the least amount of time.

1. Does the labeling work with other probes? For example, have you successfully labeled the same target using the same primary antibody and a fluorescent or enzymatic probe? If so, then you know that the target or the primary antibody to which your Nanogold® conjugate is binding is intact and working as it should. In this case, you should check the Nanogold conjugate and labeling procedure, and any reagents applied afterwards, such as silver enhancers, gold enhancers, or poststains.

2. If you are using silver or gold enhancement, are these reagents working? If you can test them in an experiment where you have used another immunogold probe that is known to be good, this will rule these out as the source of your low labeling.

3. Does the Nanogold reagent appear as it should? Nanogold conjugates in solution are a clear, pale brown to greenish-brown. If the color has changed, particularly if it is red, purple, blue, or gray, this is an indication that the Nanogold may be degraded. To be sure, check the conjugate by UV/visible spectroscopy. The UV/visible absorption spectrum of a Nanogold conjugate is shown below. Because antibodies do not absorb at wavelengths higher than about 300 nm, the region from 320 nm to 800 nm, marked in red as the Nanogold absorbance profile, will be the same for all Nanogold conjugates (note that because bovine serum albumin is added to stabilize the solution, absorbance at 280 nm will be higher for commercial Nanogold conjugates than is shown).

   

   UV/visible absorption spectrum of a Nanogold-Fab' conjugate. The region marked in red, the, "Nanogold absorbance profile," is the region in which only Nanogold absorbs: neither the antibody nor the additional bovine serum albumin added to stabilize the conjugate absorbs in this region. Therefore this profile should be similar in all Nanogold conjugates.

   If you observe a profile that is significantly different - especially the appearance of a broad maximum between 500 and 550 nm, or a significant increase in absorbance at longer wavelengths - this indicates decomposition of the Nanogold particle. Additional spectra are available in our Guide to Gold Cluster Labeling on our web site.

   If the spectrum appears to be normal, then you can check the integrity and reactivity of the Nanogold conjugate using blot tests, as described below.

4. Checking conjugate integrity: is the Nanogold still attached? You can check whether the Nanogold particle is still attached to the antibody using a simple blot test. Spot 1 µL of the Nanogold conjugate onto a nitrocellulose membrane, air-dry for 15 minutes, then develop with LI Silver (30 minutes), HQ Silver (20 minutes), GoldEnhance EM (5 minutes), or a silver enhancement reagent from another manufacturer (develop as directed for dot blots). If the Nanogold label is intact and still conjugated, you will see a brown to black spot develop. If you do not see this and you know that the silver or gold enhancement reagent is good, the Nanogold has dissociated; please contact us.

   If you obtain a strong signal in this blot, the problem may lie with the antibody part of the Nanogold conjugate. You can test this as described below.

5. Is the Nanogold conjugate binding to its target? To check whether the immunoreactivity of the Nanogold conjugate has been compromised, blot 1 µL of your primary antibody (0.1 - 1 mg/mL is a good concentration to use) onto a nitrocellulose membrane. Block with 4% bovine serum albumin (BSA) in phosphate-buffered saline (PBS), pH 7.4, for 30 minutes at 37°C, then wash with 1% BSA in PBS for 5 minutes. Incubate for one hour with a 1 : 50 dilution of the Nanogold conjugate, also in 1% BSA in PBS. Wash three times with 1% BSA in PBS, then three times with PBS, then twice with deionized water. Develop with silver or gold enhancement as described above. A brown or black spot confirms reactivity of the Nanogold conjugate towards your primary.

   If you obtain strong reactivity with this test, then you will need to revise your procedure. Try higher concentrations or primary antibody and Nanogold conjugate, longer incubation (overnight), or a higher degree of permeabilization before application; once you have achieved successful labeling, you can then gradually reduce the degree of permeabilization if you require better ultrastructural preservation.

   It should be noted that different antibodies, or antibodies with the same specificity from different suppliers, can differ in their reactivity, and we have occasionally found that antibodies we use for conjugation may be only weakly reactive towards some primaries. If this is the case, you may need to modify your labeling scheme to include a more widely used secondary, and use the Nanogold conjugate as a tertiary labeled probe.

Because they are covalently linked rather than adsorbed, Nanogold conjugates are highly stable. Provided they are not allowed to dry out or become contaminated, they can last well beyond the nominal year's shelf life if they are refrigerated at 4°C; we have tested them after two or three years and found blot detection sensitivity to be unchanged (Nanogold conjugates include low levels of a bacterial inhibitor).

Dot blots, as described above, are an excellent test for reactivity of Nanogold conjugates. For product testing purposes, we consider clear detection of 10 ng of target after silver enhancement to be acceptable (In practice, we can usually detect 0.1, and frequently 0.01 ng of target after silver enhancement), so a dot blot which develops a clear signal with 1 or 5 ng of target (or primary antibody) is a good indication of strong binding. However, the spots do become progressively lighter with smaller amounts of target, and if you compare your blots with one that shows how the spots look for a series of dilutions of target, it will give you a good qualitative idea of how good your conjugates are.

Two examples of different dot blots with Nanogold conjugates are shown below to illustrate the range of spot intensities you might encounter. In (b) the first (darkest) pair of spots (top left) contain 10 ng of target IgG; reading left to right along the top row, the second pair contains 5 ng, and the third 1 ng. If your test spots with 1 or 5 ng of target look like any of these, your conjugate is working well. After silver or gold enhancement, the spots should be black (sometimes purplish, sometimes brownish), rather than the deep red found with colloidal gold.

Our experience with conventional colloidal gold is more limited, but for conjugates of 5, 10 and 15 nm colloidal gold, the development of a clear red spot with 50 ng of target is generally a good indication that the conjugate is intact and working as it should. As shown above, you should see progressively lighter spots down to about 5 or 10 ng, sometimes 1 ng for 15 nm gold, for good preparations. For larger gold preparations, the spots will be darker. A deep cherry-red spot is a good indication of a working conjugate.

More images are available on our web site and in Hainfeld and Furuya's paper describing Nanogold, below.

For extended storage, we do not usually freeze Nanogold conjugates. If you need to do so, freezing at -20°C in 30% glycerol is recommended in order to avoid ice crystal damage.

Reference:

• Hainfeld, J. F., and Furuya, F. R.: A 1.4nm Gold cluster covalently attached to antibodies improves immunolabeling. J. Histochem. Cytochem., 40, 177-184 (1992).
  • Abstract (courtesy of JHC Online
  • Reprint (PDF) (courtesy of JHC Online

More information:

• Catalog information - Nanogold conjugates
• Nanogold-antibody conjugates - Product Information
• Nanogold-streptavidin - Product Information
• Technical help - Nanogold conjugates
• Guide to Gold Cluster Labeling
• Application: Nanogold pre-embedding labeling with HQ Silver enhancement for immunoelectron microscopy
• Nanogold and Undecagold: spectra and labeling calculation
• Nanogold - References, sorted by Application

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FluoroNanogold: Check your Labeling before EM Processing

• Increased fluorescence brightness and higher quantum yield.
• Improved solubility: lower background signal and higher signal-to-noise ratios.
• Fluorescence remains high and consistent across a wider pH range.

Since we now offer both Alexa Fluor®* 488 and Alexa Fluor®* 594 FluoroNanogold, you can now use these probes to differentiate multiple targets using different colored fluorescence.

In addition to its use for combined and correlative fluorescence and transmission electron microscopic labeling, the combined fluorescent and gold probe FluoroNanogold is useful for a number of other correlative microscopy methods. In recent articles, we have highlighted its use for correlative fluorescence and scanning electron microscopy, and for correlative optical and X-ray fluorescence microscopy. The structure of Alexa Fluor®* 488 FluoroNanogold-Fab', and some results obtained with it, are shown below.

Aggregated, highly phosphorylated tau protein is a pathological marker of Alzheimers disease (AD) and other tauopathies. Iliev and co-workers recently used FluoroNanogold as part of their ultrastructural investigations into the effects of modifications to the amino acid sequence motifs in the microtubule-binding repeats of tau. In native tau, motifs of alternating polar and apolar amino acids within the microtubule-binding repeats are interrupted by small breaking stretches. Minimal mutation of these breaking sequences yielded a unique tau mutant containing longer stretches of polar/apolar amino acids which aggregated instantly without losing its microtubule-binding capacity. Immunofluorescence and fluorescence recovery after photobleaching (FRAP) studies showed that these modifications produced rapid aggregation and cytotoxicity with accompanying occurrence of pathologic tau phosphoepitopes (AT8, AT180, AT270, AT100, Ser422, and PHF-1) and conformational epitopes (MC-1 and Alz50) in CHO cells.

Aggregation was confirmed at the ultrastructural level using FluoroNanogold for pre-embedding electron microscopic analysis of Sarkosyl extracts and CHO cells. Sarkosyl extract samples were absorbed on Formvar carbon-coated grids and counterstained with 2% (w/v) aqueous uranyl acetate. For electron microscopy on cells, the cells were fixed 24 h after transfection using 2% (w/v) formaldehyde and 0.25% (v/v) glutaraldehyde (EM grade) in PBS at pH 7.4, for 30 minutes at room temperature. Pre-embedding labeling was conducted to identify the transfected cells: cells were permeabilized with 0.1% Triton X-100, then blocked with 1% bovine serum albumin, fraction V, with 0.045% (w/v) cold water fish gelatin in PBS. Pre-embedding immunogold labeling was conducted using the anti-C-terminal tau antibody (clone T46: 1:400) as the primary, and Nanogold - and Alexa Fluor 488 - conjugated goat Fab' anti-mouse IgG, diluted 1:30 in PBS with 0.05% Triton X-100 for 1 hour. Silver enhancement of the Nanogold particles was performed using LI Silver enhancement reagent. Cells were then gently scraped in PBS, collected, and postfixed with 1% (w/v) osmium tetroxide. The fixed cells were dehydrated in a graded series of ethanol containing 0.5% uranyl acetate, then embedded in Araldite by passage through propylene oxide and propylene oxide-Araldit (1:1) solutions followed by polymerization at 60°C. Ultrathin sections were cut, mounted on Formvar-coated copper grids, and counterstained with 0.4% lead citrate. All samples were analyzed on a Zeiss EM 10B electron microscope at an accelerating voltage of 60 kV.

3PO-tau phosphorylation was also assessed in human SH-SY5Y neuroblastoma cells, which natively express several tau isoforms and represent an experimental system closer to neurons. A significant increase in the phosphorylation level of 3PO-tau was found in both CHO fibroblasts and SH-SY5Y human neuroblastoma cells, at sites that correspond to known pathological hyperphosphorylation epitopes. Similar to pathological tau in the pretangle state, toxicity appeared to occur early without the requirement for extensive fibril formation. FRAP analysis demonstrated that 3PO-tau can recruit wild type (WT)-tau protein into its aggregates: WT-tau showed a significantly decreased motility in 22% of the cells. Co-aggregation was either almost complete, or absent: most cells co-expressing 3PO-tau did not affect the diffusion properties of WT-tau-YFP (and YFP alone), even in cases where WT-tau seemed to co-localize with the aggregated 3PO-tau. This shows that 3PO-tau aggregation does not greatly affect the inherent diffusion of cytosolic proteins or act as a steric nonspecific protein trap, and show that this approach is useful for the identification of proteins that are specifically recruited to aggregated tau to mediate pathological cellular processes. This mutant protein therefore provides a novel platform for the investigation of the molecular mechanisms for toxicity and cellular behavior of pathologically aggregated tau proteins and the identification of its interaction partners.

Reference:

• Iliev, A. I.; Ganesan, S.; Bunt, G., and Wouters, F. S.: Removal of pattern-breaking sequences in microtubule binding repeats produces instantaneous tau aggregation and toxicity. J. Biol. Chem., 281, 37195-37204 (2006).
  • Abstract (courtesy of the Journal of Biological Chemistry

More information:

• Catalog information: FluoroNanogold
• Alexa Fluor 488 FluoroNanogold-Fab': Product information and instructions
• FluoroNanogold: Technical help
• FluoroNanogold: References, sorted by application