Pressure Cooker-EDTA-Assisted Antigen Retrieval

PRESSURE COOKER-EDTA-ASSISTED ANTIGEN RETRIEVAL 

Immunohistochemical staining of tonsillitis, gastric adenocarcinomas, and breast carcinomas can be obtained using MIB-1 antibody in conjunction with EDTA-NAOH solution and a pressure cooker (Kim et ah, 1999b). EDTA solution is thought to be more effective than other buffers in unmasking the epitopes, presumably because it removes (chelates) tissue-bound calcium ions. 

Deparaffinized and rehydrated tissue sections on slides are immersed in jars containing 1 mM EDTA-NaOH (pH 8.0), and the jars are placed in boiling distilled water in a stainless steel 6-liter-capacity pressure cooker with an operating pressure of 103 kPa/15psi. The pressure cooker is sealed and brought to full pressure the duration of heating is 3 min. The cooker is depressurized and cooled under running tap water for 20 min. 

The sections are treated with H202 and then incubated in the primary antibody at a dilution of 1 50. This is followed by sequential incubation in the biotinylated antimouse antibody and streptavidin-biotin-labeled complex. DAB is used for 5 min as the chromogen, and the sections are lightly counterstained with hematoxylin. Positive controls involve the use of the tissue known to express the antigen under study. Negative controls involve the replacement of the primary antibody with the diluent alone or with a non-immune serum. 

Note A 1 mM EDTA solution is easier to set at pH 8.0 when it is buffered. A sodium or potassium phosphate buffer is suitable at 0.005 mM provided the grade of the reagents is of analytical quality, i.e., the content of divalent metals is typically 0.005% or less. 

---

PRESSURE COOKER-EDTA-ASSISTED ANTIGEN RETRIEVAL... 

---