Page 254 - Prevention and Treatment of Incisional Hernia- New Techniques and Materials

Chapter 12
a biomaterial a biopsy of the abdominal wall was taken at the same place where in the other rats the mesh was implanted. In some animals, incorporation of the material was insu cient and because there was no adjacent tissue, no biopsy could be taken. For Strattice® at both time points, Sepramesh® at day 28 and C-Qur® and Omyramesh® at day 90 only 1 or 2 samples could be taken because of insu cient incorporation and therefore these conditions were excluded for analysis. Biopsies were snap-frozen in Tissue-Tekc (Sakura, Alphen, Rijn, The Netherlands) with liquid nitrogen and stored at -80oC till sectioning. Sections of 6 μm were cut on a cryostat (Leica; Davis Instruments, Vernon Hills, Illinois, USA) and stored at -80oC.
Staining
Immunohistochemistry
Frozen sections were defrosted and xed in acetone. After xation sections were washed in PBS and incubated with 10% normal goat serum (Sigma- Aldrich, St Louis, MO, USA) to block non-speci c binding. After incubation sections were washed with phosphate bu ered saline and incubated with primary antibodies against CD206 (2.5 μg/mL, Abcam, 64693, Cambridge, UK), iNOS (2 μg/mL, Abcam, 15323), CD3 (1:100, Abcam, 16669), CD68 (5 ug/ml, Acris Antibodies GmbH, BM 4000, Herford, Germany). We choose the antibiodies based on literature(4, 10, 17). Irrelevant IgG was used as a negative control. Link biotinylated goat-anti-mouse (Biogenex, HK-325-UM, Fremont, CA, USA) was used at a second antibody, Label streptavidin-AP (Biogenex, HK-321-UK) as a tertiary antibody with neufuchsin as substrate. Sections were counterstained with hematoxylin (Sigma). Lung and spleen tissue were used as a positive controls. Sections were mounted with vectamount (Vector Laboratories, Burlingame, CA).
Toluidine blue (mast cells)
Sections were defrosted and xated in acetone. After washing in demineralised water the sections were placed in a toluidine blue solution (1% Toluidin blue (Fluka (Sigma), 89640) in 50% isopropanol and 50% demineralised water) for 30 minutes at 37°C. Sections were washed for 1 minute in pure isopropanol. Sections were air-dried and mounted with vectamount (Vector Laboratories).
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