20200123-0124: Coating, transfection and ICC in culture slides (reverse transfection)

Culture slides coated with Poly-D-lysine (8 wells) –> 354632 Corning

2020.01.23 Morning:

• We transfect 4 wells of each antigen (NF140, NF186, NF155, CNTN1, Double CNTN1/CASPR1, ANO2. For 4 wells:
  • 34 µL Optimem + 1,6 µL lipo
  • 34 µL Optimem + 1,08 µg DNA
• Incubate at RT for 5 min
• Unify DNA with  Lipofectamine and incubate at RT for 30 min
• In the meantime, seed the cells –> For every well: 0,3 ml of HEK medium + the volume that correspond to 60.000 cells.
• Add 17 µL of the mix (DNA+lipo) to every well
• Incubate at 37ºC for 24h.

2020.01.24 Morning: 

• Fix with PFA 4%. Incubation at RT during 10min.
• Wash with PBS 1x and remove the chambers
• Blocking solution: Goat serum 5% or Rabbit serum 1/40 in PBS for 1h at RT.
• Remove the blocking solution

Check the transfection – ICC:  

NF140 and NF186:

1. Positive control + primary Ab (anti-NF 1 /1000) + GAH594 and GAC 488 (1/1000)
2. Positive control + GAH 594(1 /1000)
3. 19-189 + GAH 594  (1/1000)
4. Negative control + primary Ab (anti-NF 1 /1000) + GAH594 and GAC 488 (1/1000)

NF155:

1. Positive control + primary Ab (anti-NF 1 /1000) + GAH594 and GAC 488 (1/1000)
2. Positive control + GAH 594(1 /1000)
3. 19-220 + GAH 594  (1/1000)
4. Negative control + primary Ab (anti-NF 1 /1000) + GAH594 and GAC 488 (1/1000)

CNTN1:

1. Positive control + primary Ab (anti-CNTN1 1 /1000) + RAH594 and RAG488 (1/1000)
2. Positive control + RAH 594 (1 /1000)
3. 19-192 + RAH 594  (1/1000)
4. Negative control + primary Ab (anti-CNTN1 1 /1000) + RAH594 and RAG 488 (1/1000)

CNTN1/CASPR1:

1. Positive control (Piffort) + primary Ab (anti-CASPR1 1 /1000) + GAH594 and GAR 488 (1/1000). Blocked with Goat
2. Positive control (Piffort) + GAH 594 (1 /1000). Blocked with Goat
3. Positive control (Piffort) + primary Ab (anti-CNTN1 1 /1000) + RAH594 and RAG488 (1/1000). Blocked with Rabbit
4. Positive control (Piffort) + RAH 594 (1 /1000). Blocked with Rabbit

ANO2:

1. MS153 + primary Ab (anti-ANO2 1 /500) + GAH594 and GAR 488 (1/1000)
2. MS153 + GAH 594(1 /1000)
3. MS 217 + GAH 594  (1/1000)
4. Negative control + primary Ab (anti-ANO2 1 /500) + GAH594 and GAR 488 (1/1000)

RESULTS:

• Las células están bien transfectadas pero tienen una morfología un poco extraña
• Se ven perfectamente los pacientes positivos sin anticuerpo comercial (en todos los antígenos, incluído ANO2, en que se ve positivo tanto MS153 como MS217)