Page 89 - Slipped Capital Femoral Epiphysis Pathogenetic and Clinical aspects

Specimens and histomorphology
The biopsy specimens were fixed in 4% buffered formaldehyde, pH 7.5 and embedded in paraffin immediately after collection. Deparaffinized sections were routinely stained with H&E.
Immunohistochemistry.
Immunohistochemical analyses were performed on three μm thick sections from paraffin-embedded biopsies either routinely with the immunostainer of Roche
Ventana Benchmark Ultra (Tucson, USA) or applied manually. In the latter case, the
sections were deparaffinized with xylene and hydrated through grades of ethanol.
Endogenous peroxidase was blocked with 0,3% H2O2/methanol and followed by
antigen retrieval with 10 mM Tris 0,1mM EDTA pH9 (TEpH9) in a microwave for 10
minutes or with Citrate pH6 (Citr.pH6) for 20 minutes at 100°C. Tissue sections from
thyroid, placenta, ovary, tonsil and stomach were used as positive controls, as well
as Tissue Micro Arrays for androgen receptor (AR), Cytokeratin (Cam5.2), estrogen
receptor (ER) and S100. 5 Details concerning the use and detection method of the antibodies are described
in table 1.
Detection of the antibodies in the Benchmark Ultra the Optiview detectionkit including DAB was used in combination with CC1 at 100°C as antigen retrieval. Manual application detection was used with either the highly sensitive PowerVision Plus method (PV plus; Immunologic, Duiven, The Netherlands) for antibodies raised in mice or Envision anti-Mouse/Rabbit HRPTM (Dako, Glostrup, Denmark) for antibodies raised in rabbits. Visualization of the manual method Liquid DAB+ (Dako, Glostrup, Denmark) as chromogen was used and counterstained with Mayer’s hematoxylin, dehydrated with grades of ethanol, cleared with xylene. All sections were mounted with Tissue Tek ® coverslipping film (Sakura Finetek Europe B.V., Alphen aan den Rijn, The Netherlands). (see table 1)
Immunohistochemistry was scored semiquantitively, using three categories: ‘negative’(0),‘slightly positive’(0.5) and‘positive’(1). Staining patterns in chondrocytes were categorized in the nuclei and cytoplasm while simultaneously, the surrounding stromal cells in the metaphysis (in particular osteocytes) were assessed as controls in the same biopsy with similar subdivision in nuclear- and cytoplasmic patterns. All but one immunohistochemical procedures were duplicated. All biopsies were scored twice by two observers (MW and FvK).
Slipped Capital Femoral Epiphysis
87