IMMUNOCYTOCHEMICAL IDENTIFICATION OF PROLIFERATING CELLS IN VASCULAR TISSUE (ANTI-BrdU); Diaminobenzadine

IMMUNOCYTOCHEMICAL IDENTIFICATION OF PROLIFERATING CELLS IN VASCULAR TISSUE (ANTI-BrdU); Diaminobenzadine (DAB) brown detection

1. Sections of paraformaldehyde fixed, OCT-embedded vascular tissue are sectioned at 7 to 10 mm and stored, desiccated at -70°C until needed.
2. Thaw slides immediately before use, usually 10-30 min. prior to beginning the protocol.
3. Fix in acetone 5 min., air dry for approximately 20 min., RT.
4. Immerse in 0.3% H2O2/Methanol for 15 min., RT. (450µl H2O2 in 150ml Methanol).
5. Immerse in Proteinase K/ 1xPBS for 10 min., RT (7.5µl Proteinase K of 20 mg/ml stock in 150ml 1x PBS for a final concentration of 1µg/ml).
6. Rehydrate in 1x PBS twice for 5 min. each, RT.
7. Immerse in 4N HCl for 10 min., RT.
8. Immerse in 1x TBE, pH 8.4 for 5 min., RT. Check pH of buffer with pH paper to see if it is neutral.
9. Immerse in 1x PBS. Check pH of buffer after 2 min. Apply primary antibody when pH is 7.0-7.5.
10. Prepare the working dilution of the primary antibody in 1.0% crystalline-grade Bovine Serum Album (BSA) in 1x PBS. Use BrdU antibody (Dako Catalog No. M744) at 1:20 dilution. Blot off PBS and apply 150µl of working dilution. Incubate sections in a humid chamber for 1 hour, RT. Blot off antibody, wash 2 times 5 min. each in 1x PBS.
11. Prepare the working dilution of the secondary antibody (Horse anti-mouse IgG-Vector Catalog No. BA2001) in 1.0% BSA/PBS, 2.0% normal horse serum. Prepare the secondary antibody at a 1/400 dilution. Apply 150µl and incubate 30 min., RT in a humid chamber. Blot off the antibody, wash 2 times 5 min. each in 1x PBS.
12. Prepare the working dilution of ABC-Peroxidase complex or the Vector ABC-peroxidase Elite (Catalog No. PK-6100) before finishing the previous step. Mix 5ml 1x PBS, two drops of Solution A, and two drops of Solution B, and allow to sit at 4°C for 30 min. prior to use. Apply enough to cover the sections on the slide, and incubate for 1 hour. Blot off the solution, wash 2 times 5 min. each in 1x PBS, followed by 1 wash in 100mM Tris pH 8.2 for 5 min.
13. Make up substrate solution immediately before use. Add 5 ml of sterile water, two drops of buffer from Vector DAB substrate kit (Catalog No. SK-4100), four drops DAB, two drops H2O2 , and two drops of Nickel from kit, in 15ml tube (wrap the tube in foil to protect the substrate from light) . Incubate in the dark for 20-30 min., stop color reaction by blotting off substrate and rinsing in dH2O water for 5-10 min. Caution: DAB is highly carcinogenic, so handle with gloves and work on absorbent towels. Inactivate solutions by pouring into a beaker containing 3% KMnO4 and 2% Na2CO3 in water, and then dispose down sink.
14. Counterstain with hematoxylin, 1% acid-alcohol, Scott's solution, dehydrate through graded alcohols, then xylenes per protocol below, and coverslip for viewing.

    Solutions:

    Hydrogen peroxide

    SIGMA Catalog No. H-1009 (5 ml)

    Proteinase K

    Dissolve Proteinase K (Sigma Catalog No. P-4914) in dH2O for a concentration of 20mg/ml.

    1% BSA/PBS

    1g Fraction V BSA in 100ml 1x PBS. Mix in a beaker with low heat. Aliquot in 15ml tubes and store at -20°C.

    Hematoxylin

    75ml Gill's hematoxylin no.2 in 75ml dH2O.

    1% Acid-alcohol

    2ml HCl in 198ml of 70% ethanol.

    Scott's Solution

    NaHCO3, 2g

    MgSO4.7H2O, 20g

    dH2O, 1000ml

    Counterstaining Protocol:

    Hematoxylin - 10 sec.

    rinse in water until water is clear

    Very quick dip in 1% acid-alcohol (< 1 sec.)

    rinse in water

    Scott's solution - 20 sec.

    rinse in water

    Dehydrate through graded alcohols (70%, 95%, 95%, 100%, 100%)

    Xylenes

    Coverslip