Protocol revelation amplified : DBS Range

I. Required material

Reagents
- Primary antibody
- Primary Antibody Diluent
- Pre-Blocking solution
- Mounting medium

Buffers
- Immuno Wash Buffer
- Xylene or dewaxing reagents
- Absolute ethanol
- Distilled or deionized water



II. Experimentation length
- 10 mn of blocking
- 30-60 mn of sections incubation in primary antibody
- 36 or 31 mn of others incubation
- 20 mn of rinsing steps
- TOTAL : 1h31 to 2h06


III. Staining procedure

1. Peroxidase Block. Incubate slides in Peroxidase Block for 5 minutes.
Rinse slides 3 x 1 minutes with Immuno Wash Buffer.
2. Pre-Blocking (optional). Add 2 drops (100 µL) or enough volume of Pre-Blocking Solution to cover the tissue section (10 minutes).
Drain or blot off solution. Do not rinse.
3. Primary Antibody. Incubate with Primary Antibody for 30 - 60 minutes, prepared according to the manufacturer’s recommended protocol at the desired concentration. Concentrated Primary Antibodies may be diluted using Primary Antibody Diluent.
Wash slides 3 x 1 minutes in Immuno Wash Buffer.
4. Polymer Penetration Enhancer. Apply the Polymer Penetration Enhancer and incubate for 10 minutes.
Rinse slides 3 x 1 minutes with Immuno Wash Buffer.
5. PolyVue Label. Incubate the tissue with PolyVue HRP Label reagent for 10 minutes
Wash slides 3 x 1 minutes with Immuno Wash Buffer.
6. Stable DAB/Plus. Prepare the Stable DAB/Plus substrate working solution (see above).
Incubate tissue for 5-10 minutes with prepared Stable DAB/Plus substrate solution. Monitor level of staining to determine optimal time of incubation.
Rinse slides 3 x 1 minutes with water.
7. Counterstain. Incubate tissue for 1 minute with Counterstain (e.g. Hematoxylin).
Wash slides 3 x 1 minutes in water, followed 1 x 1 minute in Immuno Wash Buffer, then 1 x 1 minute in water.
8. Dehydrate&Coverslip. Dehydrate tissues through graded ethanol series, followed by xylene series.
Apply coverslips with permanent mounting medium.


IV.Search engine
- Primary antibody