Table 5.
Comparison of α-tubulin, fluorescent proteins (e.g., GFP), and fluorescent neuronal tracers (e.g., fluorescent dextran) as normalization factors
| FEATURE | METHOD
|
||
|---|---|---|---|
| α-TUBULIN | FLUORESCENT PROTEINS (E.G., GFP) | FLUORESCENT NEURONAL TRACERS (E.G., FLUORESCENT DEXTRAN) | |
| Type of standard | Internal | External | External |
| Need for genetic modification | No | Yes | No |
| Need for delivery, loading, or labeling | No | Yes, by transfection or use of transgenic mice expressing GFP. | Yes, in most cases, dyes have to be delivered to neurons. |
| Delivery/loading/labeling methods | N/A | Biological (viral vector), chemical (e.g., cell-penetrating peptides, α-toxin of S. aureus), vehicle (e.g., liposomes), mechanical (e.g., microinjection, whole-cell patch clamping, gene gun), electrical (electroporation) | Chemical (e.g., cell-penetrating peptides, α-toxin of S. aureus), vehicle (e.g., liposomes), mechanical (e.g., microinjection, whole-cell patch clamping, gene gun), electrical (electroporation) |
| Effects on neurons | N/A | Protein delivery methods may alter the physiology of neurons or cause toxicity. Chromosomal insertion by viral vectors may disrupt gene expression. | Loading/labeling neurons may alter the physiology or cause toxicity. The dyes themselves may be toxic to cells. |
| Distribution pattern | Similar to physiological distribution. | Distribution pattern may be uneven in a single neuron (e.g., puncta-like pattern, Ref. 38 and Ma, B., unpublished results). The distal-most part or fine structure may not be labeled. The pattern may be variable between cells due to different levels of protein expression (38,39). | The standards have to be anterogradely or retrogradely transported in neurons. In some cases, distribution pattern may be uneven in a single neuron (e.g., puncta-like pattern, Ref. 40). The distal-most part or fine structure may not be labeled. The pattern may be variable between cells due to different amounts of dye delivered (37). |
| Efficiency of delivery, loading, or labeling methods | N/A | Transfection efficiency may be low because primary neurons are difficult to transfect especially in older neuronal cultures. | Loading efficiency is variable. Depends on delivery methods. |
| Aldehyde-fixable | Yes | Yes, but some fluorescent proteins may become lost after fixation because GFP is highly water soluble and diffusable. | Yes, but only for specially treated dyes, e.g., lysinated dextrans. |
| Loss of fluorescence after immunofluorescent staining | No loss | Some fluorescent proteins are highly water soluble and may be lost after staining. | Fluorescent dyes may be washed out after staining. Lysinated dextrans are better. |
| Loss of fluorescence after FISH | No loss | Some fluorescent proteins are highly water soluble and might be lost after harsh treatment used in FISH. | Fluorescent dyes may be washed out after harsh treatment used in FISH. Lysinated dextrans are better. |
| Detection methods | Antibody staining | Fluorescence from fluorescent protein itself or antibody against fluorescent protein (in the case of GFP loss, see Ref. 38). | Fluorescence from conjugated fluorescent dye or antibody against the conjugated fluorescent dye or hapten. |
| Whole-cell analysis | Possible | Not possible | Not possible |
| Cell segment analysis | Possible | Possible | Possible |
| Live cell imaging and analysis | No. However, it is possible with Tubulin Tracker. | Possible | Possible |
| Prior tests before application | Need to test for constant expression in some cases (35), although it is a house-keeping gene. | Might be needed as GFP expression in different cells might be variable. | Might be needed as the amount and distribution of dyes loaded into different cells might be variable. |
| Accuracy | +++ | +++ | +++ |