Table 1.
Immunostaining Conditions
| Antibody | Animal/condition | Antigen retrieval | Quenching | Blocking | Primary | Secondary | Developing |
|---|---|---|---|---|---|---|---|
| ALDH1L1 | Stressed CX3CR1 knockout | – | 0.3% H2O2 in PBS, then 0.1% NaBH4 in PBS | 5% NGS + 0.5% gelatin in PBS | 1:1,000 in blocking buffer | 1:500 goat anti‐rabbit in blocking buffer | Incubation with Streptavidin‐HRP made in blocking, 1:1,000 |
| OLIG2 | Stressed CX3CR1 knockout | – | 0.1% NaBH4 in PBS, also 0.3% H2O2 in PBS after secondary | 10% FCS + 3% BSA in TBS | 1:1,200 in blocking buffer | 1:200 goat anti‐rabbit in TBS | Incubation with TBS ABC, 1:100 |
| IBA1 | Stressed CX3CR1 knockout | – | 0.3% H2O2 in PBS, then 0.1% NaBH4 in PBS | 10% FCS + 3% BSA, 0.01% Triton in TBS | 1:1,000 in blocking buffer | 1:300 goat anti‐rabbit in 0.01% Triton TBS | Incubation with 0.01% Triton in TBS ABC, 1:100 |
| GFP | Stressed CX3CR1 knockout | – | 0.1% NaBH4 in PBS, also 0.3% H2O2 in PBS after secondary | 10% FCS + 3% BSA in TBS | 1:5,000 in blocking buffer | 1:200 goat anti‐chicken in 0.01% Triton TBS | Incubation with TBS ABC, 1:100 |
| CD11b | Stressed CX3CR1 knockout | – | 0.3% H2O2 in PBS, then 0.1% NaBH4 in PBS | 5% NGS + 0.5% gelatin in PBS | 1:800 in blocking buffer | 1:200 goat anti‐rat in blocking buffer | Incubation with Streptavidin‐HRP made in blocking, 1:1,000 |
| P2RY12 | Stressed CX3CR1 knockout | – | 0.1% NaBH4 in PBS also 0.3% H2O2 in PBS after secondary | 10% FCS + 3% BSA in TBS | 1:500 in blocking buffer | 1:200 goat anti‐rabbit in blocking buffer | Incubation with TBS ABC, 1:100 |
| 4C12 | Susceptible animals from social defeat stress | – | 0.3% H2O2 in PBS, then 0.1% NaBH4 in PBS | 5% NGS + 0.5% gelatin in 0.05% Triton PBS | 1:50 in blocking buffer | 1:200 goat anti‐rat in blocking buffer | Incubation with Streptavidin‐HRP made in blocking, 1:1,000 |
| 4D4 | Control animals from social defeat stress | Citrate buffer @ 70°C for 40 min | 0.3% H2O2 in PBS, then 0.1% NaBH4 in PBS | 10% FCS + 3% BSA in TBS | 1:1,200 in blocking buffer | 1:300 goat anti‐rat in blocking buffer | Incubation with TBS ABC, 1:100 |
| MHCII | Stressed CX3CR1 knockout | – | 0.3% H2O2 in PBS after secondary | 10% FCS + 3% BSA in TBS | 1:500 in blocking buffer | 1:200 goat anti‐rat in blocking buffer | Incubation with TBS ABC, 1:100 |
| TREM2 | APP‐PS1 21‐months old | Citrate buffer @ 70°C for 40 min | 0.3% H2O2 in PBS, then 0.1% NaBH4 in PBS | 5% NDS + 0.5% gelatin in PBS | 1:100 in blocking buffer | 1:300 donkey anti‐sheep in blocking buffer | Incubation with Streptavidin‐HRP made in blocking, 1:1,000 |
| CD206 | Susceptible animals from social defeat stress | Citrate buffer @ 70°C for 40 min | 0.3% H2O2 in PBS, then 0.1% NaBH4 in PBS | 5% NGS + 0.5% gelatin in PBS | 1:500 in blocking buffer | 1:300 goat anti‐rat in blocking buffer | Incubation with Streptavidin‐HRP made in blocking, 1:1,000 |
| CD11c | Susceptible animals from social defeat stress | Citrate buffer @ 70°C for 40 min | 0.3% H2O2 in PBS, then 0.1% NaBH4 in PBS | 5% NGS + 0.5% gelatin in PBS + 0.01% Triton | 1:500 in blocking buffer | 1:300 goat anti‐Armenian hamster in blocking buffer | Incubation with Streptavidin‐HRP made in blocking, 1:1,000 |