Wittrup:Immunofluorescence of frozen sections

Immunofluorescence of frozen sections

Materials

primary antibody

secondary antibody

serum in which secondary antibody is raised

0.1% tween20 in PBS

Hydrophobic slide marker

Humidifier chamber (place a piece of filter paper inside a pipette tip box and fill the bottom of box with water)

Day 0

1. Air dry slides overnight.

Day 1

1. Circle around samples with pap pen.

2. Wash with PBS 3x 5mins.

(optional) Fixing. Incubate with ice cold 4% paraformaldehyde for 15 mins, followed by ice cold methanol or ethanol for 20 mins at -20C.

3. Blocking. Incubate with 5% serum in 1x PBS for 1 hr at R.T. Add enough serum to cover sample. 1ml is enough for 4 samples.

4. Primary antibody incubation. Dilute antibody in blocking solution (usually 1:50 - 1:100) and incubate overnight at 4C.

Day 2

1. Wash in PBS 3x5 mins.

From now on, work in the dark

2. Incubate with secondary antibody for 1 hr at room temp (1:200 dilution in 0.1% tween-PBS).

3. Wash in PBS 4x 3mins.

4. Mount with Vectashield.