Wittrup:Immunofluorescence of frozen sections
Immunofluorescence of frozen sections
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)
1. Air dry slides overnight.
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.
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.