Optimal immunoreactivity of keratin proteins in formalin-fixed, paraffin-embedded tissue requires preliminary trypsinization. An immunoperoxidase study of various tumours using polyclonal and monoclonal antibodies.
The effect of preliminary trypsinization on the immunoreactivity of keratin proteins in formalin-fixed, paraffin-embedded tissues of a variety of tumors (squamous cell carcinomas, adenocarcinomas, mesotheliomas, and transitional cell carcinomas) was evaluated. Three types of trypsin (Type II and Type IX porcine trypsin and Type III bovine trypsin) and varying concentrations of trypsin were assessed. Immunoreactivity of keratin proteins was determined using rabbit anti-keratin antibodies and monoclonal antibodies (combination of AE1 and AE3) and immunoperoxidase techniques. Preliminary trypsinization was mandatory for optimal immunoreactivity of keratin proteins using either polyclonal or monoclonal antibodies. Excellent results were obtained using Type II porcine trypsin at concentrations of 25 mg/dl for 30-45 min or 50 mg/dl for 20 min, at 37 degrees C. Trypsin treatment with excessive concentrations of enzyme and/or extended incubation times promoted tissue digestion and in some cases, yielded decreased immunoreactivity and altered staining patterns.
Pinkus
G S
GS
O'Connor
E M
EM
Etheridge
C L
CL
Corson
J M
JM
D000230:Adenocarcinoma / Q000378:metabolism / Q000473:pathology; D000818:Animals; D000911:Antibodies, Monoclonal; D002294:Carcinoma, Squamous Cell / Q000378:metabolism / Q000473:pathology; D002295:Carcinoma, Transitional Cell / Q000378:metabolism / Q000473:pathology; D002417:Cattle; D005260:Female; D005404:Fixatives; D005557:Formaldehyde; D006801:Humans; D007124:Immunoenzyme Techniques; D007633:Keratins / Q000032:analysis*; D008654:Mesothelioma / Q000378:metabolism / Q000473:pathology; D009363:Neoplasm Proteins / Q000032:analysis* / Q000378:metabolism; D010232:Paraffin; D013194:Staining and Labeling / Q000379:methods*; D013552:Swine; D014357:Trypsin
D000911:Antibodies, Monoclonal; D005404:Fixatives; D009363:Neoplasm Proteins; D005557:Formaldehyde; D007633:Keratins; D010232:Paraffin; D014357:Trypsin