A practical approach to routine immunostaining of paraffin sections in the microwave oven.
The Streptavidin-Biotin Complex (SABC) Immunostaining method can be carried out by performing the rinsing and blocking steps and in addition the incubation with the primary and the secondary antibody sera in the microwave oven. Irradiation of the Streptavidin-Biotin Complex reduces stain activity due to destruction of horseradish peroxidase (HRP) (Boon & Kok, 1988). Therefore, we decided not to perform this step in the microwave oven. The microwave incubations can be performed using antisera dilutions of 1:1000 (instead of 1:50) in tissue fixed with Kryofix, allowing staining in staining racks. This keeps hands-on time low and simplifies the microwave steps. It is very difficult to obtain reproducible results in the microwave oven using droplet incubations due to problems with hot spots and antenna effects. These problems are avoided when cuvettes are used and air is blown through the solutions during microwave incubations. With effective temperature control the method is highly reproducible, and takes at least 100 min less than the conventional SABC method. It is, in particular, attractive when large series of slides must be routinely immunostained and when reproducible results are desired.
Leiden Cytology and Pathology Laboratory, The Netherlands.
M E
ME
Boon
F C
FC
Hendrikse
P G
PG
Kok
P
P
Bolhuis
L P
LP
Kok
D000906:Antibodies / Q000276:immunology; D001426:Bacterial Proteins / Q000276:immunology; D001710:Biotin / Q000276:immunology; D006735:Horseradish Peroxidase; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D008198:Lymph Nodes / Q000033:anatomy & histology; D008872:Microwaves; D010232:Paraffin; D013194:Staining and Labeling; D019809:Streptavidin; D013696:Temperature; D014860:Warts / Q000473:pathology
D000906:Antibodies; D001426:Bacterial Proteins; D001710:Biotin; D010232:Paraffin; D019809:Streptavidin; D006735:Horseradish Peroxidase