2-Step Immunofluorescence Protocol:
Fresh Frozen Tissue Sections

(5-15 µm cryosections on charged (plus) slides)

Required Reagents:
Acetone
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)
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. Allow cryosections to air dry 30 minutes to 1 hour prior to fixation.
2. Actone (ice cold) 10 minutes
3. Air dry 30 minutes in hood
4. Circle section with a hydrophobic barrier pen.
5. Wash Solution - Do not allow sections to dry for the remaining procedure.
6. Ready-To-Use IHC Blocking Solution (cat# IHC-101b) - 15 minutes
7. Primary Antibody Incubation: 30 minutes 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.
8. Wash Solution - 3 changes for 5 minutes each
9. Secondary Antibody Incubation: 30 minutes 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.