(1:600) as primary antibody (Antibody Diluent Dako S 3022). The primary
antibodies were generated in our lab and have been published
previously [2]. Negative controls included antibody diluent controls
and isotype controls (Southern Biotech Mouse-anti IgG1). Rinsing
with TRIS for 2 × 5 min was done before next step.
Sections were incubated with biotinylated secondary antibody
(Antimouse IgG/Rabbit IgG) universal antibody for 30 min and avidin–
biotin horseradish peroxidase complex for 30min (Vectastain Universal
Elite ABC Kit). After each step the slideswere rinsedwith TRIS buffer for
2 × 5 min.
Peroxidase was localized in tissue sections by incubation with a
peroxidase substrate (chromogen 3-amino-9-ethylcarbazol (AEC);
Invitrogen AEC single solution) for 10 min and rinsed for 10 min
under tap water. Hematoxylin was used for counterstaining for 10 s
(Hematoxylin Gill's Formula, Merck). After clearing coverslips were
mounted with glycerol gelatin (Kaisers Glyceringelatine, Merck) and
slides were examined by brightfield microscopy by an experienced gynecological
pathologist.
A hGH-V specific mAb for immunohistochemistry could not be
found. Thus, a set-up of two antibodies (mAb 6F1 und mAb 7E8) was
established to detect hGH-V by an indirect approach: mAb 7E8 detects
hGH-N (growth hormone produced in the pituitary gland) only and
mAb 6F1 detects both hGH-N and hGH-V.