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Immunohistochemistry Protocol (Paraffin)

*IMPORTANT: See product data sheet for the appropriate wash buffer and antigen unmasking procedure.
• For Citrate/PBST, use steps 5a, 6a and C1.
• For Citrate/TBST, use steps 5b, 6a and C1.
• For EDTA/PBST, use steps 5a, 6b and C2.
• For EDTA/TBST, use steps 5b, 6b and C2.
A Solutions and Reagents
1. Xylene
2. Ethanol, anhydrous denatured, histological grade (100% and 95%)
3. Deionized water (dH2O)
4. Hematoxylin (optional)
5. *Wash Buffer:
a. For Citrate/PBST OR EDTA/PBST: 1X PBS/0.1% Tween-20 (wash buffer): To prepare 1 L add 100 ml 10X PBS to 900 ml dH2O. Add 1ml Tween-20 and mix.
10X Phosphate Buffered Saline (PBS): To prepare 1 L add 80 g sodium chloride (NaCl), 2 g potassium chloride (KCl), 14.4 g sodium phophate, dibasic (Na2HPO4) and 2.4 g potassium phosphate, monobasic (KH2PO4) to 1 L dH2O. Adjust pH to 7.4.
b. For Citrate/TBST OR EDTA/TBST: 1X TBS/0.1% Tween-20 (wash buffer): To prepare 1 L add 100 ml 10X TBS to 900 ml dH2O. Add 1 ml Tween-20 and mix.
10X Tris Buffered Saline (TBS): To prepare 1 L add 24.2 g Trizma base (C4H11NO3) and 80 g sodium chloride (NaCl) to 1 L dH2O. Adjust pH to 7.6 with concentrated HCl.
6. *Antigen Unmasking Solution:
a. For Citrate/PBST OR Citrate/TBST: 10 mM Sodium Citrate Buffer: To prepare 1 L, add 2.94 g sodium citrate trisodium salt dihydrate (C6H5Na3O7•2H2O) to 1 L dH2O. Adjust pH to 6.0.
b. For EDTA/PBST OR EDTA/TBST: 1 mM EDTA: To prepare 1 L add 0.372 g EDTA (C10H14N2O8Na2• 2H2O) to 1 L dH2O. Adjust pH to 8.0.
c. Alternative Unmasking: 10 mM Tris: To prepare 1 L add 1.21 g Tris Base (C4H11NO3) to 1 L dH2O. Adjust pH to 10.0.
7. 3% Hydrogen Peroxide: To prepare, add 10 ml 30% H2O2 to 90 ml dH2O.
8. Blocking Solution: 5% horse serum or goat serum diluted in recommended wash buffer.
9. Biotinylated secondary antibody.
10. ABC Reagent: Prepare according to manufacturer’s instructions 30 minutes before use.
11. DAB Reagent or suitable substrate: Prepare according to manufacturer’s recommendations.

B Deparaffinization/Rehydration
NOTE: Do not allow slides to dry at any time during this procedure.
NOTE: Consult product data sheet for recommended wash buffer.
1. Deparaffinize/hydrate sections:
a. Incubate sections in three washes of xylene for 5 minutes each.
b. Incubate sections in two washes of 100% ethanol for 10 minutes each.
c. Incubate sections in two washes of 95% ethanol for 10 minutes each.
2. Wash sections twice in dH2O for 5 minutes each.

C *Antigen Unmasking
NOTE: Consult product data sheet for specific recommendation for the unmasking solution.
1. For Citrate/PBST OR Citrate/TBST: Bring slides to a boil in 10 mM sodium citrate buffer pH 6.0 then maintain at a sub-boiling temperature for 10 minutes. Cool slides on bench top for 30 minutes.
2. For EDTA/PBST OR EDTA/TBST: Bring slides to a boil in 1 mM EDTA pH 8.0 followed by 15 minutes at a sub-boiling temperature. No cooling is necessary.
3. Alternate: Bring slides to a boil in 10 mM Tris pH 10.0 followed by 10 minutes at a sub boiling temperature. Cool slides on bench top for 30 minutes.

D Staining
1. Wash sections in dH2O three times for 5 minutes each.
2. Incubate sections in 3% hydrogen peroxide for 10 minutes.
3. Wash sections in dH2O twice for 5 minutes each.
NOTE: Consult product data sheet for recommended wash buffer.
4. Wash section in wash buffer for 5 minutes.
5. Block each section with 100-400 μl blocking solution for 1 hour at room temperature.
6. Remove blocking solution and add 100-400 μl diluted primary antibody to each section. (Dilute antibody in blocking solution.) Incubate overnight at 4°C.
7. Remove antibody solution and wash sections in wash buffer three times for 5 minutes each.
8. Add 100-400 μl secondary antibody, diluted in blocking solution per manufacturer’s recommendation, to each section. Incubate 30 minutes at room temperature.
9. If using ABC avidin/biotin method, make ABC reagent according to the manufacturer’s instructions and incubate solution for 30 minutes at room temperature.
10. Remove secondary antibody solution and wash sections three times with wash buffer for 5 minutes each.
11. Add 100-400 μl ABC reagent to each section and incubate for 30 minutes at room temperature.
12. Remove ABC reagent and wash sections three times in wash buffer for 5 minutes each.
13. Add 100-400 μl DAB or suitable substrate to each section and monitor staining closely.
14. As soon as the sections develop, immerse slides in dH2O.
15. If desired, counterstain sections in hematoxylin.
16. Wash sections in dH2O two times for 5 minutes each.
17. Dehydrate sections:
a. Incubate sections in 95% ethanol two times for 10 seconds each.
b. Repeat in 100% ethanol, incubating sections two times for 10 seconds each.
c. Repeat in xylene, incubating sections two times for 10 seconds each.
18. Mount coverslips.