Neuroscience Support—Troubleshooting

The Mitotracker™ dyes should be imaged soon after staining because over time, those dyes can be toxic.

Neurons are more difficult to transduce than many other cells. The main way to improve transduction is to label with a higher number of particles per cell. For primary neurons, it can also help to transduce them at the time of plating rather than on established cultures. There can also be a slower onset of expression in neurons and peak expression often occurs on day 2–3 rather than 16 hours after transduction.

DiI is a lipophilic dye that resides mostly in lipids in the cell, when cells are permeabilized with detergent or fixed using alcohol this strips away the lipid and the dye. If permeabilization is required CM-DiI can be used because this binds covalently to proteins in the membrane; some signal is lost upon fixation/permeabilization, but enough signal should be retained to make detection possible.

Since these dyes insert into lipid membranes, any disruption of the membranes leads to loss of the dye. This includes permeabilization with detergents like Triton™ X-100 or organic solvents like methanol. Permeabilization is necessary for intracellular antibody labeling, leading to loss of the dye. Instead, a reactive dye such as CFDA SE should be used to allow for covalent attachment to cellular components, thus providing for better retention upon fixation and permeabilization.

Observing both types of transport is typical for biocytin. The conjugated cholera toxin subunit B products have been observed to travel only retrogradely.

Here are our recommendations:

• Use one of our extensive selection of secondary antibodies conjugated to bright, photostable Alexa Fluor™ dyes. The degree of labeling for each conjugate is 2–8 fluorophores per IgG molecule, with potentially three secondary antibody-binding sites per primary antibody, providing signal amplification of ~10–20 fluorophores per primary antibody.
• Alternatively, primary antibody labeling can be detected with a biotinylated secondary antibody in conjunction with either a fluorescent streptavidin or a streptavidin bridge followed by a biotinylated reporter such as Qdot™ biotin. Although processing times increase with additional incubation and endogenous biotin–blocking steps, detection sensitivity also improves as a result of the labeled streptavidin.
• For low-abundance targets, signal amplification may be necessary for optimal signal-to-noise ratios. Tyramide signal amplification (TSA) is an enzyme-mediated detection method that utilizes the catalytic activity of horseradish peroxidase (HRP) to generate reactive fluorophore-labeled tyramide radicals. These short-lived tyramide radicals covalently couple to nearby residues, producing an amplified fluorescent signal localized at the HRP–target interaction site.
• For improved detection sensitivity with rapidly bleaching dyes, our SlowFade™ Diamond or ProLong™ Diamond antifade reagents have been shown to increase photostability and reduce initial fluorescence quenching in fixed cells, fixed tissues, and cell-free preparations.
• Please review this web page for further optimization tips.

• A blocking step should be performed to reduce fluorescence due to non-specific antibody binding. A common blocking step is the addition of a 2–5% solution of bovine serum albumin (fraction V defatted BSA). Another approach employs the addition of a 5–10% solution of serum from the species in which the secondary antibodies were raised. For example, when using goat anti-mouse IgG secondary antibodies, samples may be effectively blocked with 5–10% normal goat serum. To further reduce background fluorescence, the Image-iT™ FX Signal Enhancer can be included as a pre-blocking step to decrease non-specific labeling due to charge interactions between the dyes on the conjugates and the cellular constituents.
• If you are using a secondary antibody make sure that the species of the antibody is not the same as the species of the sample. For example don’t use an anti-mouse secondary antibody on mouse tissue.
• Titrate the antibody to the lowest concentration you can use and still get adequate signal.
• Try using a fluorescently tagged primary antibody because it should give reduced background but be aware this can reduce signal intensity.

If permeabilization must be performed, use dyes that covalently attach to proteins in the membrane, such as CellTracker™ CM-DiI. If permeabilization is performed with lipophilic dyes that are not covalently bound to proteins in the membrane, the dye is lost by the permeabilization detergent along with the lipids.

• Confirm that the tracer you are using crosslinks to proteins or has a primary amine for fixation—either a hydrazide, lysine fixable dextran, or a protein conjugate.
• Use aldehyde-based fixatives to cross link the amines on the tracer.
• Inject a larger amount or higher concentration of the tracer. Tracers are generally injected at 1–20% concentrations (10 mg/mL or higher).
• Confirm that you are using the correct fluorescent filter for detection. You can perform a spot test by pipetting a small amount of the undiluted stock solution of the tracer onto a slide, then view under the filter you are using on your microscope. This will confirm if the tracer fluorescence can be detected and the fluorescent microscope filter is working properly. 
• Review tissue fixation and handling procedures to confirm if any reagents or processing procedures could be affecting the tracer.

If the tracer you chose is a lipophilic dye and fix with methanol, the lipids are lost with the methanol. If you have to use methanol fixation then choose a tracer that will covalently bind to proteins in the neurons.

Ensure that the dextran you are using is the fixable form (i.e., contains a primary amine). Dextrans that do not contain a primary amine will not be fixed. Another factor could be that the concentration of the dextran is too low, and the concentration use can be increased up to 10 mg/mL.

If you want to see the most detailed structure you should use the low molecular weight conjugated dextrans such as the 3,000 MW dextrans.

If you use our FluoVolt™ Membrane Potential Kit (Cat. No. F10488), the kit provides a background suppressor to reduce this problem. For other indicators, consider the use of BackDrop™ Background Suppressor (Cat no. R37603, B10511, and B10512). 

Our NeuroTrace™ Nissl stains label the Nissl substance which is composed of ribosomal RNA associated with the rough endoplasmic reticulum and is present in high amounts in neuronal cells. These dyes are not completely specific for neurons, but will selectively stain neurons based on their high level of protein synthesis. In some cases they may show staining of other cell types such as glia, so you may need to decrease the staining concentration to obtain more selective neuronal labeling. We suggest dilutions in the range of 20- to 300-fold for neuronal staining.