Setting up the cells and basic processing is the same as for
the standard immunofluorescence protocolFixation
Add 30ul of 4% Paraformaldehyde for 30 min @ room-temperature
(RT).
Wash 1x with PBS for 5 min.
Note: During washing period always place 6 well dishes on
orbital shaker.
Permeabilization
Add 30ul of 0.2 % CHAPS in PBS for 2 min @ RT Wash 1x with PBS/ 0.02% Tween 20 for 5 min
Wash 1x with PBS/0.02% Tween 20/ 1% BSA for 5 min
Primary antibody
Add 30ul of 6A7 antiboby (dilution 1:100) in PBS/3% BSA
Incubate in a humidified chamber for 60 min @ 37ºC.
Washes
Wash with PBS/0.02% Tween 20/1% BSA for 5 min
Second antibody
6A7 activation is best observed by double staining. We use
either cytochrome c or 2G2 (human specific integral membrane
protein from the inner mitochondrial membrane) or Hsp60 (lumenal
mitochondrial marker - you can buy this from Stressgen). Our
cytochrome c antibody was raised in sheep and should be used at a
dilution of 1:750. The 2G2 antibody is a mouse monoclonal and is
used at 1:100 dilution. It can be purchased from Exalpha
Biologicals. Hsp60 depends on what Stressgen is selling at the
moment.
Secondary antibodies
From here on in the procedure is the same as the standard
protocol.
