(For use with Worksheets 7, 8 and 9)

In many cases the fixation and processing steps involved in the preparation of tissues results in loss of immunoreactivity of antigens. Often this can be reversed by the use of appropriate antigen retrieval techniques such as microwave antigen retrieval or proteolytic digestion.

Standard protocols for some of these techniques are outlined below. It should be noted that in many cases incubation times will vary depending upon the particular processing method that has been used, or for different antigens.

Note: This method provides a general procedure for use with the majority of Serotec reagents. In some cases specific recommendations are provided on product datasheets, and these method should always be used in conjunction with product and batch specific information provided with each vial. Please note that a certain level of technical skill and immunological knowledge is required for the successful design and implementation of these techniques - these are guidelines only and may need to be adjusted for particular applications.

Proteolytic antigen retrieval using Trypsin

Reagents:

Calcium Chloride 0.1g

Trypsin (Sigma Type II) 0.1g

Distilled Water 100ml

Sodium Hydroxide (0.1M)

Method:

1. Dissolve calcium chloride in distilled water and pH to 7.8 with sodium hydroxide. Store at 37^oC.
2. Dissolve trypsin in calcium chloride solution.
3. Place sections in Trypsin solution at 37^oC 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 100ml

Sodium Hydroxide (0.1M)

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 Treatment using STUF

Reagents

Serotec Target Unmasking Fluid (STUF), Cat. No. HIS003)

or

0.01M citrate buffer pH 6.0

Method:

1. Place slides in suitable container of prepared STUF/citrate buffer, cover with clingfilm (vented) and heat to 90^oC (Please note citrate buffer must boil). If the STUF/buffer evaporates after 5 minutes, top up with hot distilled water.
2. Let sections stand for 15 minutes to cool.
3. Wash in TBS and proceed with staining.