Wittrup:Immunofluorescence of frozen sections: Difference between revisions
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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. | 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 1:100) and incubate overnight at 4C. | 4. Primary antibody incubation. Dilute antibody in blocking solution (usually 1:50 - 1:100) and incubate overnight at 4C. | ||
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From now on, work in the dark | 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. | 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. | 3. Wash in PBS 4x 3mins. | ||
4. Mount with Vectashield. | 4. Mount with Vectashield. | ||
Latest revision as of 07:37, 4 November 2011
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.
