Common Immunohistochemistry (IHC) Reagent Formulations
10% Neutral Buffered Formalin (10% NBF)
4% Formaldehyde (w/v), 0.01 M Phosphate
Formulation (1 L)
| Component | Amount |
|---|---|
| 40% Formaldehyde stock solution | 100 mL |
| NaH₂PO₄·H₂O | 0.4 g |
| Na₂HPO₄ | 0.65 g |
| Deionized water | Add up to 1 L |
pH:
7.2-7.4 (adjusted with NaOH or HCl)
Storage:
1 month at room temperature; up to 3 months at 4°C
Usage:
Tissue immersion for 24 h, followed by running water rinse for 2 h
0.01 M Citrate Buffer
10 mM Trisodium Citrate, 1.8 mM Citric Acid
Formulation (500 mL)
| Component | Amount |
|---|---|
| 0.1 M Trisodium Citrate | 9 mL |
| 0.1 M Citric Acid | 41 mL |
| Deionized water | Add up to 500 mL |
pH:
6.0 ± 0.05
Storage:
1 month at 4°C
Usage:
Place sections in retrieval box, heat at 121°C for 2 min (pressure cooker) or 98°C for 10 min (microwave), then cool naturally to room temperature
3% H₂O₂ Methanol Solution
3% (v/v) H₂O₂
Formulation (10 mL, prepare fresh)
| Component | Amount |
|---|---|
| 30% H₂O₂ | 1 mL |
| Methanol | 9 mL |
Usage:
Incubate at room temperature for 10 min, rinse with PBS 3 × 3 min
Note: This reagent should be prepared fresh before use
5% Normal Serum (from same species as secondary antibody)
5% (v/v)
Formulation (1 mL)
| Component | Amount |
|---|---|
| Normal serum | 50 μL |
| PBS | 950 μL |
Usage:
Block at room temperature for 30 min, drain without washing
PBS-BT
1% BSA, 0.05% Tween-20
Formulation (100 mL)
| Component | Amount |
|---|---|
| BSA | 1 g |
| Tween-20 | 50 μL |
| PBS | Add up to 100 mL |
Storage:
≤ 1 week at 4°C; for long-term storage, aliquot and freeze at -20°C
Usage:
Dilute both primary and secondary antibodies with this solution; recommended starting concentrations: primary antibody 1:100, secondary antibody 1:200
DAB-H₂O₂ Working Solution
0.5 mg mL⁻¹ DAB, 0.003% H₂O₂
Formulation (5 mL, prepare fresh)
| Component | Amount |
|---|---|
| DAB stock solution (50 mg/100 mL TB, pH 7.6, 0.22 μm filtered) | 5 mL |
| 30% H₂O₂ | 5 μL |
Usage:
Apply to cover tissue, incubate at room temperature for 1-5 min (monitor under microscope), stop with running water
Note: This reagent should be prepared fresh before use
Harris Hematoxylin
0.4% Hematoxylin (w/v), 0.08% Mercuric Oxide
Formulation (500 mL)
| Component | Amount |
|---|---|
| Hematoxylin | 2 g |
| Absolute ethanol | 20 mL |
| Potassium aluminum sulfate | 40 g |
| Deionized water | 480 mL |
| Mercuric oxide | 1 g (add slowly with thorough stirring) |
Preparation:
Filter, let stand for maturation for 24 h
Storage:
6 months at room temperature in brown bottle
Usage:
Dilute working solution 1:1 with deionized water, stain nuclei for 30 s-1 min, blue with running water for 5 min
Buffers
PBS (0.01 M, pH 7.4)
| Component | Amount (1 L) |
|---|---|
| NaCl | 8.0 g |
| KCl | 0.2 g |
| Na₂HPO₄·12H₂O | 2.9 g |
| KH₂PO₄ | 0.2 g |
| Deionized water | 1 L |
Preparation:
Autoclave, store at room temperature
TBS-T (0.05 M Tris, 0.15 M NaCl, 0.05% Tween-20, pH 7.6)
| Component | Amount (1 L) |
|---|---|
| Tris base | 6.05 g |
| NaCl | 8.8 g |
| Deionized water | 900 mL → adjust pH to 7.6 → add up to 1 L |
| Tween-20 | 0.5 mL |
Mounting Medium
Neutral Balsam
Usage:
After dehydration through graded ethanol series (70% → 80% → 95% → 100% ×2, 2 min each) and clearing with xylene (2 × 3 min), apply appropriate amount of neutral balsam and cover with coverslip
| Jeff Jeff is a pathology product expert at EnkiLife, specializing in immunohistochemistry research and product development for over 12 years. He has a high level of insight into user needs and is committed to helping our clients conduct immunohistochemistry experiments and accelerate scientific discoveries. |
