Table 1.
IHC Protocols for Validated Cellular Markers in Pig and Human Tissues.
| Marker | Primary Antibody | Antigen Retrieval | Secondary Reagents |
|---|---|---|---|
| MBP | Anti-MBP Polyclonal Antibody (LS-C312288, LifeSpan BioSciences, Inc., Seattle, WA) in Diluent; Pig:1:600/1 hr; Human: 1:200/1 hr | HIER, Citrate buffer pH 6.0, 110C for 15 min; 20 min cool down | Dako EnVision+ System- HRP Labeled Polymer Anti-rabbit, 30 min (Dako North America, Inc., Carpentaria, CA) |
| Anti-Calbindin-D28K | Anti-Calbindin-D-28K Monoclonal (C9848, Sigma-Aldrich Co. LLC, St. Louis, MO) in Diluent; Pig: 1:6000/1 hr Human: 1:6000/1 hr |
HIER, Citrate buffer pH 6.0, 110C for 15 min; 20 min cool down | Dako EnVision+ System- HRP Labeled Polymer Anti-mouse, 30 min (Dako North America, Inc., Carpentaria, CA) |
| GFAP | Anti-GFAP Polyclonal Antibody (AB16997, Abcam, Plc., Cambridge, MA) in Diluent; Pig: 1:200/1 hr Human: 1:200/1 hr |
HIER, Citrate buffer pH 6.0, 125C for 5 min; 20 min cool down | Dako EnVision+ System- HRP Labeled Polymer Anti-rabbit, 15 min (Dako North America, Inc., Carpentaria, CA) |
| AIF1 | Anti-AIF1 Polyclonal (#019-19741, Wako Pure Chemical Industries, Ltd., Richmond, VA) in Diluent; Pig: 1:2000/1 hr Human: 1:1000/1 hr | HIER, Citrate buffer pH 6.0, 125C for 5 min; 20 min cool down | Dako EnVision+ System- HRP Labeled Polymer Anti-rabbit, 30 min (Dako North America, Inc., Carpentaria, CA) |
| S100 | Anti-S100 Polyclonal (#Z0311, Dako North America, Inc., Carpentaria, CA) in Diluent; Pig: 1:4000/45 min Human: 1:4000/45 min |
HIER, Citrate buffer pH 6.0, 110C for 15 min; 20 min cool down | Dako EnVision+ System- HRP Labeled Polymer Anti-rabbit, 20 min (Dako North America, Inc., Carpentaria, CA) |
| beta-Tubulin III | Anti-beta-Tubulin III Polyclonal (#ab18207, Abcam, Plc., Cambridge, MA) in Diluent; Pig: 1:500/1.5 hr/37C oven Human: 1:500/1.5 hr/37C oven | HIER, Citrate buffer pH 6.0, 110C for 15 min; 20 min cool down | Dako EnVision+ System- HRP Labeled Polymer Anti-rabbit, 30 min (Dako North America, Inc., Carpentaria, CA) |
| vWF | Anti-vWF Polyclonal (A0082, Dako North America, Inc., Carpentaria, CA) in Diluent; Pig: 1:500/1 hr Human: 1:4000/1 hr | Dako Proteinase K, 5 min (Dako North America, Inc., Carpentaria, CA) | Dako EnVision+ System- HRP Labeled Polymer Anti-rabbit, 15 min (Dako North America, Inc., Carpentaria, CA) |
| Cleaved Caspase-3 | Anti-Cleaved Caspase-3 Polyclonal (#9661, Cell Signaling Company, Danvers, MA) in Diluent; Pig: 1:200/1 hr Human: 1:200/1 hr | HIER, Steamer, Citrate Buffer pH 6.0, incubate for 40 min, 20 min cool down | Dako EnVision+ System- HRP Labeled Polymer Anti-rabbit, 30 min (Dako North America, Inc., Carpentaria, CA) |
| PCNA | Anti-PCNA Monoclonal (Clone PC10, #M0879, Dako North America, Inc., Carpentaria, CA) in Diluent; Pig: 1:4000/1 hr Human: 1:4000/1 hr | HIER, Citrate buffer pH 6.0, 125C for 5 min; 20 min cool down | Dako EnVision+ System- HRP Labeled Polymer Anti-mouse, 15 min (Dako North America, Inc., Carpentaria, CA) |
| EMA | Anti-EMA Polyclonal Antibody (LS-C30532, LifeSpan BioSciences, Inc., Seattle, WA) in Diluent; Pig: 1:400/1 hr Human: 1:400/1 hr | HIER, Citrate buffer pH 6.0, 110C for 15 min; 20 min cool down | Dako EnVision+ System- HRP Labeled Polymer Anti-rabbit, 30 min (Dako North America, Inc., Carpentaria, CA) |
| Glucose transporter 1 (Glut1) | Anti-Glut1 Polyclonal Antibody (LS-C188008, LifeSpan BioSciences, Inc., Seattle, WA) in Diluent; Pig: 1:250/1 hr Human: 1:250/1 hr | HIER, Citrate buffer pH 6.0, 110C for 25 min; 20 min cool down | Dako EnVision+ System- HRP Labeled Polymer Anti-rabbit, 30 min (Dako North America, Inc., Carpentaria, CA) |
| Laminin | Anti-Laminin Polyclonal Antibody (#ab11575, Abcam, Plc., Cambridge, MA) in Diluent; Pig: 1:100/2 hr Human: 1:100/2 hr | Pig—HIER, Tris pH 9.0, 125C for 5 min; 20 min cool down Human-Dako Proteinase K, 5 min (Dako North America, Inc., Carpentaria, CA) |
Dako EnVision+ System- HRP Labeled Polymer Anti-rabbit, 15 min (Dako North America, Inc., Carpentaria, CA) |
| CD20 | Anti-CD20 Polyclonal (ACR 3004 A, B, Biocare Medical, Pacheco, CA) in Diluent; Pig: 1:200/1 hr; Human: 1:200/1 hr | HIER, Citrate buffer pH 6.0, 110C for 15 min; 20 min cool down | Dako EnVision+ System- HRP Labeled Polymer Anti-rabbit, 30 min (Dako North America, Inc., Carpentaria, CA) |
| CD13 | Anti-CD13 SP182 (AM33219PU-N, Acris Antibodies GmbH, San Diego, CA) in Diluent; Pig: 1:1200/1 hr Human: 1:1200/1 hr | HIER, Citrate buffer pH 6.0, 110C for 15 min; 20 min cool down | Dako EnVision+ System- HRP Labeled Polymer Anti-rabbit, 30 min (Dako North America, Inc., Carpentaria, CA) |
| Vimentin | Anti-Vimentin Monoclonal (#M0725, Dako North America, Inc., Carpentaria, CA) in Diluent; Pig: 1:300/15 hr Human: 1:300/15 hr | HIER, Citrate buffer pH 6.0, 110C for 15 min; 20 min cool down | Dako EnVision+ System- HRP Labeled Polymer Anti-mouse, 15 min (Dako North America, Inc., Carpentaria, CA) |
| Desmin | Anti-Desmin Monoclonal (#M0760, Dako North America, Inc., Carpentaria, CA) in Diluent; Pig: 1:100/15 min Human: 1:100/15 min | HIER, Citrate buffer pH 6.0, 110C for 15 min; 20 min cool down | Dako EnVision+ System- HRP Labeled Polymer Anti-mouse, 15 min (Dako North America, Inc., Carpentaria, CA) |
| Myoglobin | Anti-Myoglobin Polyclonal Antibody (#A324, Dako North America, Inc., Carpentaria, CA) in Diluent; Pig: 1:1600/2 hr Human: 1:25,600/2 hr | None | Dako EnVision+ System- HRP Labeled Polymer Anti-rabbit, 15 min (Dako North America, Inc., Carpentaria, CA) |
Abbreviations: MBP, Myelin Basic Protein; HIER, Heat Induced Epitope Retrieval (unless otherwise specified—Decloaking Chamber Plus, Biocare Medical, Concord, CA); HRP = horseradish peroxidase; “Diluent” is, unless otherwise specified, Dako Antibody Diluent (Dako North America, Inc., Carpentaria, CA); GFAP, glial fibrillary acidic protein; AIF1, allograft inflammatory factor 1; vWF, von Willebrand Factor; PCNA, proliferating cell nuclear antigen; EMA, epithelial membrane antigen.