Table 4.
Summary table of antigens, antibodies, and corresponding immunolabeling detection performance based on fixation procedures.
| Comparing distribution patterns of FGF14 and selected AIS proteins and neuronal markers using different options of tissue fixation | ||||
|---|---|---|---|---|
| Target protein | Antibody: host, dilution and source | Fresh-frozen Option A | Perfusion-fixed Option B | Freshly prepared acute brain slices Option C |
| Protein immunoreactivity was highly detected using | ||||
| FGF14 | Mouse anti-FGF14 (1:300, NeuroMabs, catalog number 75-096) | * and ** Prominent staining in AIS (Figures 1B, 2C–F; Supplementary Figure 1A) | ++, ++++, and ± Prominent staining in AIS and soma (Figures 3B,D, 4–8; Supplementary Figures 2–6) | # Prominent staining in AIS and soma (Figures 9A–C) |
| Ankyrin-G | Mouse anti-Ankyrin-G (1:1000, NeuroMabs, catalog number 75-146) | Not tested | ++, +++, and ± Prominent staining in AIS (Figures 6, 7C; Supplementary Figures 3, 5C, 6C,D) | # Prominent staining in AIS (Figures 9A–D) |
| Ankyrin-G | Mouse anti-Ankyrin-G (1:300, NeuroMabs, catalog number 75-147) | * Prominent staining in AIS ‡ | ++ and +++ Prominent staining in AIS | # Prominent staining in AIS |
| β-IV-spectrin | Chicken anti-β-IV-spectrin (1:500, gift from Dr. M. Komada, Tokyo Institute of Technology, Tokyo, Japan) | * Prominent staining in AIS ‡ (Figure 2D) | ++, +++, ++++, and ± Prominent staining in AIS (Supplementary Figures 6A,B) | Not tested |
| Parvalbumin | Rabbit anti-Parvalbumin (1:1000, Abcam catalog number Ab11427) | ** weak staining in the soma (Figure 2F) | ++ and ++++ Prominent staining of somata, dendrites, and AIS; +++ Prominent staining of somata and dendrites (Figures 3H–I, 4) | Not tested |
| NeuN | Guinea pig anti-NeuN (1:250, Synaptic System, catalog number 266 004) | Not tested | ++, +++, and ± Prominent staining of somata (Figures 3G, 6, 7C; Supplementary Figures 3, 6A) | # Prominent staining of somata (Figure 9D) |
| Calbindin | Rabbit anti-calbindin (1:10,000, Swant, catalog number CB38) | Not detectable | ++, +++, ++++, and +++++ Prominent staining of somata and dendrites (Figures 1A, 3F,J, 5; Supplementary Figures 5A,B) | Not tested |
| Calretinin | Mouse anti-calretinin (1:3000, Swant, catalog number 6B3) | Not tested | +++ Prominent staining of somata and dendrites (Figure 1A) | Not tested |
| Sox2 | Rabbit anti-Sox2 (1:1200, Millipore, catalog number AB5603) | Not tested | ++ and +++ Prominent staining of somata (Figure 7A) | Not tested |
| DCX | Goat anti-DCX (1:400, Santa Cruz Biotechnology, catalog number sc-8066) | Not detectable | ++ and +++ Prominent staining of somata and dendrites (Figure 7B) | Not tested |
| BrdU | Rat anti-BrdU (1:1000, Abcam, catalog number ab6326) | Not tested | ++ and +++ with (DNA denaturation protocol) Prominent staining of somata (Figure 7C) | Not tested |
| PanNav | Rabbit anti-PanNav (1:300, Alomone Labs, catalog number ASC-003) | * Prominent staining in soma and AIS ‡ (Supplementary Figure 1A) | ++ Prominent staining of somata, and weak detection in the AIS ‡ (Supplementary Figure 4A) | # Prominent staining in the soma |
| PanNav | Rabbit anti-PanNav (1:300, Sigma-Aldrich, catalog number S6936) | * (Wildburger et al., 2015; Shavkunov et al., 2013) | ++ Prominent staining of somata, and weak detection in the AIS ‡ (Figure 8A) | # Prominent staining in the soma |
| PanNav | Mouse anti-PanNav clone K58/35 (1:300, Sigma-Aldrich, catalog number S8809) | Not tested | ++ Weak detection in the AIS; ± Prominent staining in AIS (Supplementary Figures 6A,B, 7A,B) | Not tested |
| Nav1.1 | Rabbit anti-Nav1.1 (1:500, Alomone Labs, catalog number ASC-001) | * Prominent in soma and weak in AIS ‡ | ++ Prominent staining of somata, and weak detection in the AIS (Figure 8B) | # Prominent staining in the soma and weak in AIS |
| Nav1.1 | Mouse anti-Nav1.1 (1:500, NeuroMab, catalog number 75-023) | Not tested | ++ Prominent staining of somata, and weak detection in the AIS | Not tested |
| Nav1.2 | Rabbit anti-Nav1.2 (1:300, Alomone Labs, catalog number ASC-002) | * weak detection ‡ | ++ Prominent staining in AIS of the neurons in the cerebellum ‡ (Figure 8C; Supplementary Figure 4C) | Not tested |
| Nav1.2 | Mouse anti-Nav1.2 (1:300, NeuroMab, catalog number 75-024) | * Wildburger et al., 2015 | ++ weak detection ‡ (Supplementary Figure 4B) | # weak detection |
| Nav1.6 | Rabbit anti-Nav1.6 (1:300, Alomone Labs, catalog number ASC-009) | * Prominent staining in AIS ‡ | ++ and ± Prominent staining in AIS and Node of Ranvier (Figure 8D; Supplementary Figures 4D, 7A,B) | # Prominent staining in AIS (Figures 9A–D) |
| Nav1.6 | Mouse anti-Nav1.6 (1:300, NeuroMab, catalog number 75-026) | Not tested | ++ Prominent staining in AIS ‡ | # Prominent staining in AIS |
| Caspr | Mouse anti-Caspr (1:500, NeuroMab, catalog number 75-001) | Not tested | ++ Prominent staining of the Node of Ranvier ‡ (Supplementary Figure 4D) | Not tested |
| MAP2 | Mouse anti-MAP2 (1:500, Novus Biologicals, catalog number NBP2-25156) | Not tested | ++ Prominent staining of somata and dendrites ‡ (Supplementary Figure 5C) | Not tested |
| MAP2 | Chicken anti-MAP2 (1:500, Synaptic System, catalog number 188 006) | Not tested | ++ Prominent staining of somata and dendrites ‡ (Supplementary Figures 5A,B) | Not tested |
Protein immunoreactivity was detected using the following fixation and post-fixation procedures:
Fresh-frozen sections immersed in acetone (7 min) (Shavkunov et al., 2013; Wildburger et al., 2015).
Fresh-frozen sections immersed in acetone (7 min) followed by methanol (7 min).
Perfusion-fixed tissue: animal was perfused intracardially with commercially available 1% formaldehyde + %0.5 methanol (Master-Tech Scientific), then sections immersed in acetone (7 min).
Perfusion-fixed tissue: animal was perfused intracardially with 4% PFA.
Perfusion-fixed tissue: animal was perfused intracardially with 4% PFA, and then sections immersed in acetone (7 min).
Perfusion-fixed tissue: animal was perfused intracardially with commercially available Optimal FixTM (Master-Tech Scientific), and then sections immersed in acetone (7 min).
Brains were extracted then immersed in 4% PFA for 30 min followed by incubation in 20–30% sucrose overnight. Then sections were permeabilized with 1 % Triton X-100, 0.5 % Tween in 1X PBS for 7–10 min.
Acute brain slices: animal was perfused intracardially with ACSF, and then slices immersed in commercially available 1% formaldehyde + 0.5 methanol (Master-Tech Scientific) (30 min), followed by overnight incubation in 20–30% sucrose, then sections immersed in acetone (7 min).
Only one condition tested.