Open Access
Table 4
Details of reagents required for Immunostaining.
| Reagent Type | Reagent constitution | Reagent Quantity (calculated) |
|---|---|---|
| Wash buffer | TBS + 0.025% Triton X-100 | 250 µL (0.25 mL) Triton X-100 in 1000 mL TBS |
| Permeabilization reagent | 0.2% Triton X-100 in PBS | 20 µL (0.02 mL) Triton X-100 in 10 mL TBS |
| Fixative | 4% paraformaldehyde in PBS | 10 mL of 16% PFA in 30 mL PBS |
| Antibody diluent buffer | TBS + 1% BSA | 0.1 g BSA in 10 mL TBS |
| Blocking Buffer | 10% normal serum + 1% BSA + TBS | 1mL normal serum + 0.1 g BSA + 9 mL TBS |
| For double staining: 5% normal serum of species in which first secondary antibodies were raised + 5% normal serum of species in which second type of secondary antibodies was raised + 1% BSA + TBS | ||
| Note: Goat and donkey serum 60 mg/mL – Jackson Immuno Research | ||
| Buffers | 1× TBS (10× TBS comprised of 500 mM tris base + 1.5 M NaCl + HCl to set pH to 7.4); 1× PBS | 100 mL of 10× in 900 mL distilled deionized H2O |
BSA is bovine serum albumin, TBS is tris-buffered saline, PBS is phosphate-buffered saline.
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