How to Assemble a Micro NC DAB Kit in 6 Simple Steps

A micro NC (non-capture) DAB (Diaminobenzidine) kit stains tissue samples for microscopy. To assemble it, prepare reagents in order, apply to slides, and incubate properly. The process takes ~90 minutes with key steps for blocking, primary/secondary antibody binding, and DAB chromogen development. Always follow safety protocols for chemical handling.

Required Materials

• Micro NC DAB kit (includes chromogen, substrate buffer, hydrogen peroxide)
• Primary and secondary antibodies (species-specific)
• Phosphate-buffered saline (PBS) or Tris-buffered saline (TBS)
• Blocking serum (normal serum from secondary antibody host species)
• Microscope slides with tissue sections (deparaffinized/hydrated)
• Humidified staining chamber
• Hydrophobic barrier pen
• Distilled water and wash bottles
• Timer and pipettes

Step-by-Step Assembly Instructions

1. Slide Preparation

1. Deparaffinize and hydrate tissue sections through xylene and graded alcohols.
2. Circle sections with a hydrophobic pen to contain reagents.
3. Rinse slides in PBS/TBS buffer for 5 minutes.

2. Blocking Endogenous Peroxidase

1. Mix 3% hydrogen peroxide (from kit) with methanol (1:10 ratio).
2. Apply to slides for 10-15 minutes in the dark.
3. Rinse 3× with PBS/TBS (2 minutes each).

3. Blocking Non-Specific Binding

1. Apply blocking serum (diluted 1:5 in PBS/TBS) for 20 minutes.
2. Do not rinse-tap off excess serum before adding primary antibody.

4. Primary Antibody Incubation

1. Dilute primary antibody in blocking serum (follow datasheet ratios).
2. Apply to slides and incubate in a humid chamber for 60 minutes at room temperature (or overnight at 4°C).
3. Rinse 3× with PBS/TBS (3 minutes each).

5. Secondary Antibody and DAB Development

1. Apply biotinylated secondary antibody (diluted per kit instructions) for 30 minutes.
2. Rinse 3× with PBS/TBS.
3. Mix DAB chromogen (1 drop) + substrate buffer (1 mL) + hydrogen peroxide (1 drop from kit).
4. Apply DAB solution to slides for 2-10 minutes (monitor brown color development under microscope).
5. Stop reaction by rinsing in distilled water for 5 minutes.

6. Counterstaining and Mounting

1. Counterstain nuclei with hematoxylin (30 seconds to 1 minute).
2. Dehydrate slides through graded alcohols and xylene.
3. Mount with permanent mounting medium and coverslip.

Troubleshooting Common Issues

Safety Precautions

• DAB is a potential carcinogen-wear gloves, lab coat, and goggles.
• Work in a fume hood when handling powdered DAB.
• Dispose of waste according to hazardous material protocols.
• Avoid skin contact with hydrogen peroxide and organic solvents.

Time and Cost Comparison of Staining Methods