2-Step Immunofluorescence Protocol:
Formalin-fixed, Paraffin-embedded tissues and cell blocks

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

Bethyl Laboratories IHC Reagents are strongly recommended for best results

Required Reagents:
Xylene
Ethanol- histology grade
Methanol
Hydrophobic barrier pen
Concentrated IHC Wash Solution (Bethyl cat# IHC-101e)
Ready-To-Use IHC Blocking Solution (Bethyl cat# IHC-101b)
Ready-To-Use IHC Antibody Diluent (Bethyl cat# IHC-101c)
Goat anti-Rabbit IgG Antibody-FITC (Bethyl cat# A120-201F)
Fluorescent mounting media
Coverglass

Preparation of Reagents:

Wash Solution
5 ml Concentrated IHC Wash Solution (cat# IHC-101e)
995 ml dH20
Store at 4 C, expiration 3 months

Secondary Antibody
20 µl Goat anti-Rabbit IgG Antibody-FITC (Bethyl cat# A120-201F)
2 ml Ready-To-Use Antibody Diluent (cat# IHC-101c)
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% EtOH - 3 changes for 5 minutes each
3. dH20 rinse 1 minute
4. Circle section with a hydrophobic barrier pen. Do not allow sections to dry for the remaining procedure.
5. Wash Solution rinse
6. Ready-To-Use IHC Blocking Solution (cat# IHC-101b) - 15 minutes
7. Primary Antibody Incubation: 1 hour room temperature. Prepare primary antibody with Ready-To-Use IHC Antibody Diluent (Bethyl cat# IHC-101c). Optimal working dilutions should be determined experimentally by the investigator. For Bethyl IHC Antibodies: 1:100 1:500
8. Wash Solution - 3 changes for 5 minutes each
9. Secondary Antibody Incubation: 1 hour room temperature. Protect from light.
10. Wash Solution - 3 changes for 5 minutes each
11. dH20 rinse
12. Mount with fluorescent mounting media and coverslip. Use fluorescent mounting media with DAPI if counterstaining is desired.

NOTES:
This procedure is suitable for Phospho-specific Antibodies