Principle :
Cells treated with DNA damaging agents are incubated for sufficient time for PARP to become activated (e.g. for 30 min when oxidative stress is used).
In order to prevent catabolism of the polymer by poly(ADP-ribose) glycohydrolase (PARG), cells must be fixed in TCA.
Poly(ADP-ribose) is detected with monoclonal anti-poly(ADP-ribose) primary antibody. Signal is amplified by the Vector Elite mouse ABC kit containing : (i) rabbit serum ; (ii) biotinylated rabbit anti mouse immunoglobulin ; (iii) A and B reagents (for avidin biotin peroxidase complex = ABC). Peroxidase reaction is developed by cobalt enhanced Ni-DAB substrate.
The same procedure is used to detect PAR in frozen tissue sections.
Material :
10% chilled TCA (trichloracetic acid)
Blocking serum (usually a 5% serum from the host of the secondary antibody)
Anti-poly(ADP-ribose) monoclonal antibody (e.g. from Alexis Biochemicals)
Vector™ Elite Mouse ABC kit
DAB (3,3’-diaminobenzidine) Warning : highly toxic, potentially carcinogenic ! Wear protections while handling.
Xylene
Solutions :
PBS
2.56 g Na2HPO4*2H2O
10.6 g NaH2PO4
72 g NaCl
Dissolve in 1 L of distilled water : adjust to pH 7.2
PBST
2.62 g Na2HPO4*2H2O
11.5 g NaH2PO4
9 g NaCl
2-3 ml Triton X-100
Dissolve in 1 L of distilled water ; pH 7.4
Acetate buffer
16.4 g Na acetate (anhydrous)
Dissolve in 1 L of distilled water. Carefully adjust the pH to 6.0 as the buffer has very low buffer capacity !
Tris-Cobalt
1.2 g Tris
1g cobalt chloride
Dissolve in 200 mL of distilled water ; pH 7.2
DAB solution
4.7 mg DAB Warning : wear gloves and protection mask !
80 mg NaCl
100 mg NiSO4
Dissolve in 10 ml of acetate buffer. Right before use, add 1.3 ml H2O2.
Protocol description :
1. Fix your slides in 10% chilled TCA for 10 min (TCA inactivates PARG).
2. Block endogenous peroxidase activity by incubating slides in 0,5% H2O2-methanol (e.g. 177 ml methanol + 3 ml 30 % H2O2) for 15 min at room temperature (this step can be omitted if fluorescence detection is used instead of peroxidase).
3. Clear in PBS (4 x 5 min).
4. Block non-specific binding by incubating slides in blocking serum (usually 5% serum from the host of the secondary antibody, e.g. 5 % rabbit serum when using biotinylated rabbit anti-mouse Ig as secondary antibody ; for details see inlay of Vector kit). Incubate 2 hours at room temperature or 1 hour at 37°C (from this point, make sure that the slides don't dry out !)
5. Apply primary antibody (e.g. monoclonal anti-poly-ADP-ribose antibody from Alexis Biochemicals used at a: 1:300 dilution) for 1 hour at room temperature (the slides can be covered with Parafilm™ and should be kept under humid conditions).
6. Wash 5 times in PBST.
7. Apply the secondary biotinylated antibody (supplied with the Vector Elite Mouse kit) and incubate for 30 min at room temperature (the Vector™ A and B solutions should be prepared at this step, as it has to be incubated at room temperature for 30 minutes prior to use. For details, check the inlay of the Vector™ kit.)
8. Wash 5 times in PBST.
9. Apply the Vector™ A-B solution and incubate for 30 min at room temperature.
10. Wash 5 times in PBST.
11. Briefly rinse in acetate buffer.
12. Apply DAB solution and incubate at room temperature for 4 min.
13. Apply Tris-Cobalt solution and incubate at room temperature for 4 min.
14. Wash in distilled water.
15. Dehydrate.
16. Wash once in 70 % alcohol, twice in 95 % alcohol, three times in 100 % alcohol, three times in xylene (use a fumehood !)
17. Cover and archive.
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