RE: Mitochondrial immunostaining

From:Abizar Lakdawalla

RE: Mitochondrial immunostaining

bcl-2 is a protein that is located in the mitochondria and I know that there are quite a few good antibodies out there (including the ones we provide). In most paraffin sections the staining will appear as "cytoplasmic" with colorimetric end-points (BCIP/NBT gives the highest resolution as compared to AEC or DAB). To see at higher resolution you will need to stick to fluorescent end-points.

Hope this does not muddy the waters even more,

> -----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
> about
> antibodies to stain mitochondria in paraffin embedded and/or frozen
> sections
> of human lung.
> I would like to reproduce the specific stain I see using the
> histochemical
> method with an immunofluorescence technique (I'll do confocal
> microscopy
> on
> 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.
> Now
> 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
> ask
> 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
> antibody
> 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
> (2%serum/PBS
> 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,
> in
> 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
> antibody
> 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
> to
> retrieve antigens by boiling the sections in 10mM Na citrate pH 6.00,
> but
> 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,
> I'll
> greatly appreciate any help!
> Sorry for have been taking so much time of yours,
> Alessandra
> Alessandra Livraghi, PhD
> Cystic Fibrosis Center- UNC Chapel Hill
> 7013 Thurston Bowles Bldg.
> CB#7248
> Chapel Hill NC 27599
> tel: 919-966-7045
> fax: 919-966-7524

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