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

See the 2-step immunofluorescence protocol to be used for the epitope retrieval method for formalin-fixed, paraffin-embedded tissues and cell blocks. Includes required reagents, preparation steps and procedure.

Optimizing this protocol for your tissue or target? Our spatial biology team can help with antibody selection, validation, and troubleshooting.

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For antibodies made in Rabbits

Use 3-5µm paraffin sections on charged (plus) slides

Materials

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.


Normal 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 preservative.


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# DyLight 488 (A120-101D2), DyLight 550 (A120-101D3), DyLight 554 (A120-101D4), or cross-adsorbed DyLight 488 (A120-201D2), DyLight 550 (A120-201D3), DyLight 554 (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
  13. 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.
  14. Wash Solution - 3 changes for 5 minutes each
  15. Secondary Antibody Incubation: 1 hour - room temperature. Protect from light.
  16. Wash Solution - 3 changes for 5 minutes each
  17. Mount with fluorescent mounting media and coverslip. Use fluorescent mounting media with DAPI if counterstaining is desired. Protect from light.

NOTE: Suitable for Phospho-specific Antibodies