Immunohistochemistry Protocol-Cytokine Antibodies

Introduction Immunohistochemistry is used to identify the location and distribution of target antigens in cells or tissues by staining with a specific antibody. The antibody is conjugated to either a fluorescent or colorimetric label, and the location of the label seen through a microscope approximates the position of the target antigen. We present here a method for staining of cervical tissue. Purpose Procedure for indirect staining of cytokines in paraformaldehyde fixed cryostat sections with monoclonal antibodies. Procedure from Walid Al-Saleh Thesis (ULG, Pr. Boniver Service of Anatomo-pathology), Liege, Belgium. The cytokines IL-2, IL-4, IL-6, IL-10, IL-12, IFN- γ and TNF-α proteins in cervical biopsies were detected by an original immunohistology technique. Materials and Equipment • Frozen sections of sample tissues • Refrigerator (4ºC) • Glass slides • Primary antibody • Coverslips • Secondary antibody • 4% Paraformaldehyde, pH 7.4 • Hematoxylin • 1% Goat Serum in TBS/Saponin •Ethanol • Diaminobenzidine (DAB) substrate solution • Microscope • Tris-buffered Saline, 0.1% Saponin, pH 7.4 • Tris-buffered Saline/0.3% H2O2/0.1% Saponin, 0.02% NaN3 • Avidin/Biotin/Peroxidase (Vectastain, Vector Labs) Protocol 1. Dry frozen tissue sections of 8 μm thickness at room temperature for 2 hours. 2. Fix sections with 4% paraformaldehyde for 15 minutes at room temperature. 3. Wash slides 2X, 4 minutes each, with TBS/0.1% saponin. 4. Block endogenous peroxidase by incubating 30 minutes in TBS/0.3% H2O2/0.1% Saponin, 0.02% NaN3. 5. Wash slides 3X, 3 minutes each, with TBS/saponin. 6. Block non-specific binding sites with 1/100 diluted goat serum in TBS/saponin for 20 minutes. 7. Incubate overnight at 4ºC, with the appropriate antibody. 8. Wash slides 4 times in TBS/saponin, incubate the slides with biotinylated secondary antibody for 30 minutes. 9. Add avidin-biotin-peroxidase reagents, and reveal the resulting peroxidase activity by incubating the slides with a 0.5 mg/mL HRP substrate solution (DAB + H2O2 prepared in distilled water). 11. Counterstain for 1 minute with hematoxylin. 12. Dehydrate slides with sequential ethanol washes of 1 minute each starting with 75%, followed by 80% and finishing with a 100% ethanol wash. 13. Seal slides. 14. Analyze by optical microscopy. Note: It is not necessary to add detergent to the TBS buffer. The protocol can be followed s is with saponin omitted from all buffers when staining CDs

上一篇：IHC Protocol for Free Floating Brain Sections   下一篇：IMMUNOHISTOCHEMISTRY on PARAFFIN OR CRYOSECTIONS