Immunofluorescence with

polyclonal rabbit anti-EEA1 antibody # 243, bleed3

Fixation

A) PFA-fixation, Triton permeabilisation after fixation

PFA stocks in the -20°C freezer are 3% PFA in PBS. Dilute 16% PFA stock from TAAB in PBS.

1. Three rinses with PBS

2. 3%PFA in PBS for 15min.

3. Three rinses with PBS

4. 50mM ammonium chloride in PBS for 20min

5. Rinse three times with PBS. Coverslips may be stored over night at this stage

6. Before starting with the IF protocol, permeabilise 3 min with 0.2% Triton/PBS

B)Saponin permeabilisation prior to fixation

filter all saponin solutions, do not store for more than a day

Pipes-MTSB: 80 mM Pipes, 1 mM MgCl2, 5 mM EGTA; pH 6.8-7.4/KOH

1. rinse cells 3x with PBS

2. permeabilse for 5 min with 0.2% saponin in Pipes-MTSB 3. fix immediately for 15 min in 3% PFA/PBS

4. remove PFA and dispose in closed container

5. immediately add PBS and rinse 3x

!!Beware not to let the cells dry out!!!

6. replace PBS with 50 mM NH4Cl in PBS for 20 min (quench)

7. rinse 3x with PBS

8. before starting with the IF protocol, wash 5 min in 0.2% saponin/PBS

c) Methanol fixation does not work for this antibody!

Staining

Goat serum and Moviol stocks should be in the -20°C freezer G08 (IF box). Secondary antibody in the fridge.

1. 10% goat serum/PBS for 20min: BLOCKING

2. Primary antibody anti-EEA1 rabbit 293 bleed 3 in 5%goat serum/PBS at 1:1000. Spin antibody dilutions before use (1 min at full speed). Incubate coverslips face-down on 80-100 µl drops of antibody-dilution in a wet chamber for 20-30min.

4. Wash 3x 5 min with PBS.

5. Secondary antibody: anti-rabbit with Oregon-Green or TexasRed tag in 5% goat serum/PBS at 1:500. Spin antibody dilutions before use (1 min at full speed). Incubate coverslips face-down on 80-100 µl drops of antibody dilution in a wet chamber for 20-30min.

6. Wash 3x 5 min with PBS.

7. Dip into PBS and then into water.

8. Remove excess water and mount immediately on slides using ~50µl of Mowiol per 22x22mm coverslip.