2-Step Immunofluorescence Protocol: Epitope Retrieval Method for Formalin-fixed, Paraffin-embedded tissues and cell blocks

Materials:

• Xylene
• Reagent Alcohol - histology grade
• Hydrophobic barrier pen
• Epitope Retrieval Buffer-Citrate Buffer pH 6.0
• Epitope Retrieval Buffer-Tris Buffer pH 9.0
• Wash Solution
• Normal Goat Serum Blocking Solution
• IHC Antibody Diluent
• Goat anti-Rabbit IgG Antibody-DyLight conjugated (Bethyl cat# A120-101D2, A120-101D3, A120-101D4, or cross-adsorbed antibodies A120-201D2, A120-201D3, A120-201D4)
• Fluorescent mounting media with or without DAPI
• Coverglass

Preparation of Materials:

10mM Citrate Buffer pH 6.0

2.94 g Tri-sodium Citrate (dihydrate)
Volume to 1 Liter with distilled water
Check pH (pH is usually at 6.0).
Store at room temperature for 3 months or at 4 °C for 6 months.

Tris-EDTA Buffer pH9.0 (10mM Tris Base, 1mM EDTA)

1.21 g Tris Base
0.37 g EDTA
Volume to 1 Liter with distilled water
Check pH (pH is usually at 6.0).
Store at room temperature for 3 months or at 4 C for 6 months.

Norml Goat Serum Blocking Solution

20 ml normal goat serum (Filter sterilize with 0.2 µM filter)
Volume to 100 ml with PBS or TBS.
Store 2-8 °C, discard after 3 months.

IHC Antibody Diluent (50mM TBS, 1% BSA)

190 ml 1X TBS
2 g BSA (Sigma A9647)
Volume to 200 ml with TBS.
Store 2-8 °C, discard after 1 year.

*May want to use a perservative.

Wash solution

Can be any of the following: PBS, TBS or 0.01% Tween 20 in dH20. TBS wash solution is recommended for phospho antibodies.

Secondary Antibody

Goat anti-Rabbit IgG Antibody-Dylight conjugated. (Bethyl cat# A120-101D2, A120-101D3, A120-101D4, or cross-adsorbed antibodies A120-201D2, A120-201D3, A120-201D4) Dilute 1:100 with IHC antibody diluent. Prepare just prior to use. Protect from light. Optimal working dilutions should be determined experimentally by the investigator.

Procedure:

1. Xylene - 3 changes for 5 minutes each
2. 100% Reagent Alcohol - 3 changes for 5 minutes each
3. dH20 rinse
4. Place prepared Epitope Retrieval Buffer (refer to Bethyl antibody datasheet for appropriate epitope retrieval buffer) into steamer or water bath and heat to 96-100 °C
5. Add rack of slides to hot Epitope Retrieval Buffer - 20 minutes
6. Remove container and slides from steamer or water bath and cool on bench - 20 minutes
7. Slowly add dH20 for 5 minutes
8. dH20 rinse - 1 minute
9. Circle section with a hydrophobic barrier pen. Do not allow sections to dry for the remaining procedure.
10. Wash Solution - 3 changes for 5 minutes each
11. Normal Goat Serum Blocking Solution - 15 minutes
12. Primary Antibody Incubation: 1 hour - room temperature
    Prepare primary antibody with IHC Antibody Diluent
    Optimal working dilutions should be determined experimentally by the investigator. Refer to Bethyl Antibody datasheet for suggested dilution range.
13. Wash Solution - 3 changes for 5 minutes each
14. Secondary Antibody Incubation: 1 hour - room temperature. Protect from light.
15. Wash Solution - 3 changes for 5 minutes each
16. Mount with fluorescent mounting media and coverslip. Use fluorescent mounting media with DAPI if counterstaining is desired. Protect from light.

NOTE: This procedure is suitable for Phospho-specific Antibodies