RE: Mitochondrial immunostaining

From:"Caldas, Hugo"


From what you say I guess your problem probably is the secondary antibody,
as your control is staining just the same without a primary. 
When I have had this before I pre-absorbed the antibody to cells of the
tissue I am trying to stain (in your case human lung cells, if you can, or
if not you could start with a human cell line).
If this is not the problem, have you ruled out tissue autofluorescence?

Hugo Caldas
Columbus Children's Hospital
Columbus, OH

-----Original Message-----
From: Alessandra Livraghi
To: HistoNet Server
Sent: 10/24/2002 5:59 PM
Subject: Mitochondrial immunostaining

Hallo everybody!
Here I am again. Some time ago I was asking for some histochemical
method to
label mitochondria in  human tissues...I got some good advices and the
staining with SDH and NBT works pretty well.
Now I'm back on the same issue, but I'm looking for some GOOD advices
antibodies to stain mitochondria in paraffin embedded and/or frozen
of human lung.
I would like to reproduce the specific stain I see using the
method with an immunofluorescence technique (I'll do confocal microscopy
these samples)
For immunofluorescence staining, I've tryed three different antibodies
(mHSP70 from Affinity Bioreagents, Cyt b from SantaCruz and anti-OxPhos
Complex V inhibitor protein from Molecular Probes), with poor results.
I'm wondering if I'm doing something really wrong (fixation, incubations
time..)...or if it's just bad luck with antibodies.
I've just came back from the last unsuccessful staining. So I decided to
for some help from people with more experience than me!
These are the protocols I'm using:

Fixation and permeabilization: 4%PFA, 4 min RT. Wash in PBS. 100%EtOH, 2
min -20C. Wash in PBS.
Blocking: 20% serum (matched with the species where the secondary
is raisen), 3 hrs RT
Washing: 3x5min in PBS
Primary antibody: dil 1:100 or 1:50 in 2%serum/PBS, overnight 4C
Usually I include a control incubated without primary antibody
alone) and another one incubated with the same concentration of IgG
(ChromPure from JacksonLab) as my antibody (isotypic control).
Washing: 3x5min in PBS
Secondary antibody: Alexafluor 568 dil 1:1000 in 2%serum/PBS, 30 min RT,
the dark
Washing: 3x5min in PBS
Mounting with Vectashield Mounting medium (with DAPI)
Results: my isotypic control is stained exactly as the antibody treated
sample(maybe a little bit less, but nothing significant) !

PARAFFIN EMBEDDED SECTIONS (fixed in 10% NBF overnight)
Dewaxing through xilene (2x5 min) and EtOH 100% (2x3 min), 95%(2x3 min),
70%(1x3 min)
Rehydration: PBS, 30 min
Permeabilization: 0.1%Triton in PBS, 10 min RT. Wash in PBS.
Blocking: 20% serum (matched with the species where the secondary
is raisen), 3 hrs RT
From now on, everything is the same as in the protocol for frozen
sections...including the results! Actually, in this case I've tried also
retrieve antigens by boiling the sections in 10mM Na citrate pH 6.00,
nothing changed in the final results.

This is the status of my "art". I'm really disappointed!
If somebody  has a direct experience in immunostaining of mitochondria,
greatly appreciate any help!
Sorry for have been taking so much time of yours,

Alessandra Livraghi, PhD
Cystic Fibrosis Center- UNC Chapel Hill
7013 Thurston Bowles Bldg.
Chapel Hill NC 27599
tel: 919-966-7045
fax: 919-966-7524

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