ICC on FLOT1/2 transfected HEKs on MS patients

OBJECTIVES

To determine the existence of anti-FLOT1/2 antibodies in a subset MS patients.

SAMPLES

1. 128 (suspected positive using the EUROIMMUN FLOT1/2 kit)
2. 158 (suspected positive using the EUROIMMUN FLOT1/2 kit)
3. 163 (suspected positive using the EUROIMMUN FLOT1/2 kit)
4. Negative control

Each of them were performed 4 times: 2 with Triton-based permeabilization, and 2 with acetone-based permeabilization, each of them with and without primary antibodies.

MATERIALS

• Human FLOT1/2-transfected HEKs 293 grown in coverslips. Out of them:
  • Half were fixed with PFA 4%, permeabilized with Triton 0,3% and blocked with GOAT serum 5% in PBS.
  • Half were permeabilized with acetone
• PBS.
• Blocking: GOAT 5% in PBS.
• Patient’s sera diluted 1/100 with GOAT 5% in PBS.
• Primary Ab: Rat anti-FLOT1 (1/100 dillution).
• Secondary Abs: GAH 594 (1/1000) + GAR 488 (1/1000).
• Vectashield mounting medium.

PROCEDURE

• FLOT1/2-transfected HEKs were grown, permeabilized and fixed on coverslips and frozen at -80ºC. They where thawed before the ICC with Goat Serum 5%.
• Added patient’s sera at 1/10 dilution in Goat Serum 5% and incubated 1h at RT.
• Washed 3 times with PBS for the ones permeabilized with Triton and PBS-Tween for those permeabilzed with acetone (in this and subsequent cleaning steps the ones using acetone were immersed in OBS-Tween for 5 minutes while on a rotary shaker)
• (Skipped for the ones not using primary antibodies) of the Added primary commercial antibody at 1/100 dilution and incubated 1h at RT.
• (Skipped for the ones not using primary antibodies) Washed 3 times with PBS for the ones permeabilized with Triton and PBS-Tween for those permeabilzed with acetone.
• Added secondary Abs 1/1000 each and incubated 1h at RT.
• Washed 3 times with PBS and distilled water for the ones permeabilized with Triton and PBS-Tween for those permeabilzed with acetone.
• Added Vectashield mounting medium and put onto microscope slides.
• The slides were observed with a fluorescense microscope to determine wether or not the patients’ sera presented anti-FLOT1/2 antibodies.

RESULTS

All the results showed a highly intense background fluorescence that made every cell look the same, making it impossible to determine the transfected ones. With Triton permeabilization and no primary antibody, the result was still faint as in last attempts (not so on the acetone-permeabilized ones).

We can then determine that the previous faint results when not using primary antibodies were proabably not due to a lack of permeability but to another unknown factor.

To solve this problem some tests could be tried, such as using lower concentrations of serum (dillution was 1/10 in this one) or doing shorter incubations.