Ronna Hertzano – based on the Kelley lab
last updated 4-28-2003
Whole organ immuno (this example is for a polyclonal antibody made
in rabbit – e.g. MyoVI (Hasson), MyoVIIa, Pou4f3 etc)
1. Rinse the tissue in at least 1 ml PBS-0.5% tween20 for 1 hour at RT with gentle
agitation1 (tween20 concentrations can be reduced to 0.2%).
the tween20 can be substituted with triton x-100 (0.5%).
***** this protocol continues using PBST as the diluents buffer. However, after
step 1 – you can continue the entire procedure in PBS, if you wish.
2. Incubate for 1 hour in PBS-0.5% tween20 with 5% goat serum at RT with gentle
agitation (alternatively, add 1 or 3% BSA, up the goat serum to 10% or combine –
not for the above mentioned antibodies though). I did most of my immunos with a
blocking solution of PBS-0.5% triton + 10% NGS. Or 5% NGS + 3% BSA.
In some cases you might need to do antigen retrieval, specifically for nuclear
proteins.
3. Remove serum and without rinsing add your primary antibody at the correct
dilution. For whole organ immunos, it is usually better to incubate with gently
agitation at 40c o/n2.
4. Take the antibody off and put it back in a tube for re-use.
5. Rinse 3X20min at RT with gentle agitation in at least 1ml of PBS-0.5% tween20
each time.
Fluorescence detection
6. Incubate with secondary antibody diluted 1:1000 in PBS-0.5% tween20 for 1 hour
at RT, with gentle agitation. This incubation should be done in the dark (cover
the Eppendorf tube with aluminum foil or put the tube in a drawer), and the
tissue should be protected from light from this moment on.
 For staining actin, add phalloidin at the correct concentration to the
secondary antibody incubation, or it can be added as a separate 45 min
step followed by 3X20min rinses afterwards.
7. Rinse 3X20min at RT with gentle agitation in at least 1ml of PBS-0.5% tween20
each time.
8. Mount with anti-fade solution.
1
This step is usually done with the dissected cochleas placed in an Eppendorf tube, as most of the rest of
the protocol is. Be careful not to 'suck up' the cochleas during the rinses. You can put a regular tip at the
end of a transfer pipette or leave some liquid at the end with the cochleas, as throughout the protocol there
are enough rinses to get rid of undesired background
2
this step too, as the rest of the protocol is done in an Eppendorf tube.
Ronna Hertzano – based on the Kelley lab
last updated 4-28-2003
Peroxidase detection
*** for the peroxidase staining, incubate the tissue for 20min in a 0.3% solution of
H2O2 (in PBS-0.5% tween20) at RT before step 1. then rinse for 10min with PBS-0.5%
tween20 before step 1. *****again – the protocol can be done with PBS (no tween or
triton) providing an initial incubation with tween or triton was performed****
6. Add biotinylated secondary antibody (at the correct concentration) for 1 hour at
RT with gentle agitation.
7. Rinse 3X20min at RT with gentle agitation in at least 1ml of PBS-0.5% tween20.
8. Prepare the ABC complex from the peroxidase Vecstain ABC Elite standard kit,
Vector labs (do not confuse with AP = alkaline phosphatase) 30 min in advance
at RT (= add to 5ml of PBS-0.5% tween20 2 drops of solution A, mix, then 2
drops of solution B, mix and nutate/let stand for 30min).
9. Incubate the tissue with the ABC complex solution, for 1 hour at RT with gentle
agitation.
10. Rinse 3X20min at RT with gentle agitation in at least 1ml of PBS-0.5% tween20.
11. Prepare the DAB solution with the DAB reagent (to 5ml of distilled water add 2
drops of buffer stock solution and mix well, add 4 drops of DAB stock solution
and mix well, add 2 drops of the hydrogen peroxide solution and mix well and if a
grey-black stain is desired, add 2 drops of nickel solution and mix well3), Vector
labs – immediately before use and protect from light. Filter the solution at 0.8µm
(a small filter attached to a syringe), and add it to the tissue. Check under
microscope for developing color (a color can develop as fast as within a few
seconds and up to 15min).
12. Stop the reaction by adding H2O, then change again for PBS. Do not mount with
anti-fade (or color reaction will disappear). Mount with glycerol (9:1 dilution).
3
Other substrates can be bought for other colors e.g. red, green, blue etc.
Ronna Hertzano – based on the Kelley lab
last updated 4-28-2003
List of supplies

PBS

Tween20

Goat serum
Vector S-1000; cat # M1127 20ml.

anti-rabbit alexa 594 (red)
Molecular probes (www.probes.com)
Alexa Fluor 594
goat anti-rabbit IgG (H+L)
2mg/ml; 0.5ml; A-11037
** same but in green 488 – A-11034

phalloidin
Molecular probes
Alexa Fluor 488 phalloidin – A-12379

anti-fade kit
Molecular probes
SlowFade light antifade kit
S-7461

Biotinylated goat anti-rabbit
Biotinylated anti rabbit IgG (H+L) made in goat
Vector BA-1000

ABC kit
Vectastain ABC kit – elite PK-6100 standard.
www.vectorlabs.com

DAB staining
Peroxidase substrate kit DAB – SK-4100

For using monoclonal antibodies on mouse tissue use the MOM kit (Vector)