ICC on NF155 transfected HEKs on Multiple Sclerosis patients (CSF samples 1-11)

OBJECTIVES

To determine the existence of anti-NF155 antibodies in a subset MS patients.

SAMPLES

1. 038-12. Dolores Grau Fuste

2. 038-17. Montserrat Huguet Olle

3. 088-24. Jose Mosquera Gayoso

4. 114-04. Inmaculada Aranda Romero

5. 130-05. Carmen Fabra Arques

6. 131-22. Rosa M. Olucha Marine

7. 131-24. M. Torres Martínez

8. 134-14. Antonio Díaz Blanco

9. 134-18. M.Carmen Peñalba Blázquez

10. 134-20. Sonia Egido Moreno

11. 134-22. Miguel Ángel García Faxa

12. Negative control (Edu serum)

13. Positive control (15-195)

MATERIALS

• Human NF155-transfected HEKs 293 grown in coverslips. Fixed with PFA 4% and blocked with GOAT serum 5% in PBS.

• PBS.

• Blocking: GOAT 5% in PBS.

• Patient’s CSF diluted 1/1 with GOAT 5% in PBS. Control sera diluted 1/100 with GOAT 5% in PBS.

• Primary Ab: Chicken anti-NF155: AF3235 (1/1000).

• Secondary Abs: GAH 594 (1/1000) + GAC 488 (1/1000).

• Vectashield mounting medium.

PROCEDURE

• NF155-transfected HEKs were grown and fixed on coverslips and frozen at -80ºC. They where thawed before the ICC with Goat Serum 5%.

• Added patient’s CSF at 1/1 dilution in Goat Serum 5% and incubated 1h at RT. Control sera were added at 1/100 dilution.

• Washed 3 times with water.

• Added primary commercial anti-NF155 Ab: AF3235 at 1/1000 dilution and incubated 1h at RT.

• Washed 3 times with water.

• Added secondary Abs: GAH 594 and GAC 488 1/1000 each and incubated 1h at RT.

• Washed 3 times with water.

• Added Vectashield mounting medium and put onto microscope slides.

• The slides were observed with a fluorescense microscope to determine wether or not the patients’ CSF presented anti-NF155 antibodies.

RESULTS

1. Negative.

2. Negative.

3. Negative.

4. Negative.

5. Negative.

6. Negative.

7. Negative.

8. Negative.

9. Negative.

10. Negative.

11. Negative.

12. Negative.

13. Positive.