Double color immunohistochemistry
The slides were subjected to double color immunohistochemistry for CK and ZP immunoreactivity (CK/ZP). The slides were first immunostained for CK of epithelial cells and TA fibroblasts, but without hematoxylin counterstain and dehydration. Briefly, specimens were washed with phosphate buffered saline (PBS) and incubated 20 minutes with mouse-anti human CK 18, clone CY-90 (Sigma) or CK 5, 6, 8, 17, clone MNF116 (Dako Corporation, Carpinteria, CA, USA) (5 μg/ml in PBS). After extensive washing in PBS, specimens were incubated 20 minutes with swine anti-mouse IgG peroxidase conjugate (SwAM/Px; SEVAPHARMA, Prague, Czech Republic – kindly provided by Dr. Jana Peknicova, Department of Biology and Biochemistry of Fertilization, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic) diluted 1:50 and preabsorbed with rat kidney homogenate [
57]. Antigen-antibody complexes were detected by a standard diaminobenzidine technique (brown color).
After three washes in PBS, the slides were incubated for 20 minutes with rabbit antibody to heat-solubilized porcine zona (HSPZ) [
58,
59], diluted 1:20, or sheep antibody to heat-solubilized rabbit zona (HSRZ) protein [
59] (1:20), or PS1 monoclonal antibody to the meiotically expressed porcine oocyte ZP carbohydrate antigen [
38] (1:100). The antibodies were kindly donated by Dr. Bonnie S. Dunbar, Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, USA. Additional rabbit anti-HSPZ antibodies [
58] were kindly provided by Dr. Satish K. Gupta, National Institute of Immunology, New Delhi, India. The following monoclonal primary antibodies were also utilized at IgG concentration 5 μg/ml: CD31 of endothelial cells, clone JC/70A (Dako Corporation, Carpinteria, CA, USA), HLA-DR of endothelial cells and activated tissue macrophages, clone MEM-12 [
60] (Drs. Ivan Hilgert and Vaclav Horejsi, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic and Faculty of Sciences, Charles University, Prague, Czech Republic), MAPK (pan-ERK), clone 16 (BD Transduction Laboratories, San Diego, CA, USA), and Thy-1 differentiation protein of fibroblasts and pericytes, clone F15-42-01 [
61] (Dr. Rosemarie Dalchau, Institute of Child Health, University of London, London, UK).
Primary antibodies were followed by a relevant peroxidase-coupled secondary antibody – AffiniPure Goat Fab' Anti-Rabbit IgG (H+L), Abs. human serum (Protos Immunoresearch, Burlingame, CA, USA) and AffiniPure Rabbit Anti-Sheep IgG, Fc Fragment Specific – minimal cross-reaction to Human Serum Proteins (Jackson Immunoresearch Laboratories, West Grove, PA, USA), or SwAM/Px (SEVAPHARMA) as above. Antigen-antibody complexes were detected by a Vector SG detection kit according to the supplier's manual (Vector Laboratories, Inc., Burlingame, CA, USA), giving the substrate dark blue color. This contrasted with the brown color resulting from the first sequence of immunoreagents (CK immunostaining). The slides were dehydrated and mounted, without hematoxylin counterstain. A control procedure consisted of double color immunohistochemistry as above, but both primary antibodies were replaced with PBS. Additional controls included replacement of primary antibodies with nonimmune hybridoma ascites, antibodies not reacting with human tissues (mouse-anti rat Thy-1 differentiation protein, clone OX-7 – donated by Dr. Allan F. Williams, MRC Cellular Immunology Unit, Sir William Dunn School of Pathology, University of Oxford, Oxford, UK), and normal rabbit IgG.
Since the diaminobenzidine reaction product masks the antigen and catalytic sites of the first sequence of immunoreagents, preventing interaction with the reagents of the second sequence [
62], a possibility of co-expression in the same cell types was investigated. Therefore, another set of slides was similarly processed, with the opposite order of primary antibodies (ZP/CK). In addition, two sets of slides were immunostained for CK or ZP expression alone, followed by hematoxylin counterstain.
Triple color immunohistochemistry
Double color immunohistochemistry was performed as above and complemented with the third primary antibody and corresponding secondary antibody incubation. Antigen-antibody complexes were detected by a Vector VIP detection kit according to the supplier's manual (Vector Laboratories), giving the substrate a purple color. Control staining consisted of the same procedure, but primary antibodies were replaced as indicated above.
Differential interference contrast images were captured with a DEI-470 CCD Video Camera System (Optronics Engineering, Goleta, CA) with detail enhancement and CG-7 color frame grabber (Scion Corporation, Frederick, MD) supported by Scion Image public software developed at the National Institutes of Health (Wayne Rasband, NIH, Bethesda, MD). Captured images were compiled using Microsoft® Power-Point® 97 SR-2 (Microsoft Corporation, Redmont, WA) and Microsoft Photo Editor 3.0 (Microsoft Corporation).