（共享）抗原修复方法

不错的抗原修复方法，大家共享。

Proteolytic antigen retrieval using Trypsin

Reagents:

Calcium Chloride 0.1g

Trypsin (Sigma Type II) 0.1g

Distilled Water 100 ml

Sodium Hydroxide (0.1 M)

Method:

1.Dissolve calcium chloride in distilled water and pH to 7.8 with sodium hydroxide. Store at 37oC.

2. Dissolve trypsin in calcium chloride solution.

3. Place sections in Trypsin solution at 37oC and incubate for pre-determined optimum time (approximately 20-30 minutes).

4. Wash in TBS and proceed with staining.

Proteolytic antigen retrieval using pronase

Reagents:

Calcium Chloride 0.1g

Pronase 0.1g

Distilled Water 100 ml

Sodium Hydroxide (0.1 M)

Method:

1. Dissolve calcium chloride in distilled water.

2. Dissolve pronase in calcium chloride solution and pH to 7.8 with sodium hydroxide.

3. Place sections in pronase solution at room temperature and incubate for pre-determined optimum time (approximately 10 minutes).

4. Wash in TBS and proceed with staining

Heat mediated antigen retrieval

Heat mediated antigen retrieval may be used in many cases to enable staining of certain antigens in paraffin embedded tissue sections, or in some cases to improve results when staining is weak or inconsistent.

A number of buffers may be used for this purpose. The choice of buffer should be as recommended on product specific datasheets, or determined experimentally by the customer. Serotec offer a range of pre-prepared antigen retrieval buffers, or alternatively customers may prefer to prepare their own buffers using the recipes provided below.

Reagents

Antigen retrieval buffer

Method:

1. Place slides in suitable container of prepared buffer, cover with cling-film (vented) and heat to 90oC (Please note buffer must boil). Keep at this temperature for 10 minutes

2. Let sections stand for 15 minutes to cool.

3. Wash in TBS/tween and proceed with staining.

Note: Actual heating times may vary depending on the antibody being used and the fixation processes that have been utilised.

Solutions used:

TBS (Tris buffered saline) (stock solution x10 concentrated)

Sodium chloride 87.66 g

Tris 60.55 g

Distilled water 1 litre

Adjust pH to 7.4 using concentrated HCl.

TBS/Tween

Working strength TBS
1 litre

Tween 20
0.5 ml

0.01M Citrate buffer for antigen retrieval

Anhydrous citric acid
3.84 g

Distilled water
1800 ml

Adjust pH to 6.0 using concentrated NaOH.

Make up to 2 litres with distilled water

0.001M EDTA buffer for antigen retrieval

EDTA
0.37 g

Distilled water
1000 ml

Adjust pH to 8.0 with 1 M NaOH