Bethyl will be closing at 1pm on Friday, December 21st for the Christmas holidays. We will resume normal business hours on Wednesday, January 2nd.

Protocols

 

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

 

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

Required Reagents:

Preparation of Reagents:

Normal Goat Serum Blocking Solution
20 ml normal goat serum (filter sterilize with 0.2 µM filter)
Volume to 100 ml of PBS or TBS
Store at 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 add 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. Circle section with a hydrophobic barrier pen. Do not allow sections to dry for the remaining procedure.
  5. Wash Solution - 3 changes for 5 minutes each
  6. Normal Goat Serum Blocking Solution - 15 minutes
  7. 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.
  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. 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