Re: [Histonet] mouse spleen confocal

From:"Scott Parker"

Thanks for the tips. We haven't even got to the stage where we dilute
primaries. With or without antibodies added i see exactly the same thing.
Just lots of auto-fluorescence! I'm happy to try the tips you gave me, but
to be honest my primary concern at this time is to try to get a section that
isn't completely red. Any ideas on that?

On 18/04/06, Gayle Callis  wrote:
> Scott,
> Is your secondary adsorbed to mouse tissue?  You blocking serum should be
> matched to the host of the secondary and you can add 1 to 2% mouse serum
> to
> this normal serum block AND use this NSB as the diluent of the
> secondary.  The secondary should be F(ab')2 frag of IgG to not bind to fc
> receptors on your tissues.  If your secondary is made in rabbit, then you
> may get background aka autofluorescence problems when the rabbit IgG
> sticks
> to everything.
> Did you do a dilution panel on your primary starting at 10 ug/ml?  You did
> not give a concentration in ug/ml.
> I suggest you try this method to get rid of secondary entirely.
> NSB 30 min
> Strepavidin/biotin block (kit from Vector) kit instructions
> Biotinylated Rat anti Mouse CD8 (Ly3.2) diluted in the normal serum block
> I
> just described incubated for 30 min.  CD8 is an antibody that requires a
> higher concentration at times - so do a dilution panel.  Immunologist here
> indicated it was probably due to low affinity.
> Come back with Strepavidin-Alexa 633 diluted 1:1000 in buffer with Tween
> (yes, you can use it but we like a lower concentration!)
> Just to let you know, the GFP will probably NOT survive the acetone
> fixation, a common problem and CD8 will NOT survive formalin or
> paraformaldehyde fixation.  GFP is nicely lit up by using an AntiGFP
> antibody.  Rockland has an excellent Goat antiGFP for this purpose and we
> come back with donkey antiGoat f(ab')2 frag of IgG, adsorbed to mouse, a
> FITC conjugate - you will have to work faster with this one OR you can
> conjugate a favorite secondary with Alexa 488 per Molecular Probes kit -
> easy to do.
> We use Tween 20 for all our double immunofluorescence staining, just use
> 0.025%, very dilute to keep frozen sections intact.
> An excellent fixation protocol for murine CD markers for IFA work
> (including CLSM) is air dry your sections OVERNIGHT at RT, fix in 75%
> acetone/25% absolute ethanol for 5 min at RT the next day, go directly to
> buffer, do NOT AIR DRY AGAIN after fixation.   You may be surprised at
> quality of CD8 staining but beware, GFP does NOT survive this solvent
> fixation.
> t 10:04 AM 4/18/2006, you wrote:
> >Dear all,
> >Can anybody help me with some antibody staining I have been doing on
> mouse
> >spleen? We are trying to detect CD8 in 5 um sections using CD8b.2 (ly-3.2)
> (
> >53-5.8) with secondary Alexa Fluor 633 from mol. probes. Unfortunately
> all
> >we see is vast amounts of auto-fluorescence making antibody detection
> >impossible. We really need to get this sorted because ultimatley we'd
> like
> >to have GFP and two different secondaries (labelling 3 things in total).
> >Our protocol is basically:
> >
> >Spleens are removed to OCT and frozen in isopentane, sectioned (5 um) and
> >stored at -20.
> >When ready for use slides are warmed to room temp and fixed in -20 C
> acetone
> >for 3 mins then re-hydrated in PBS for 5 mins.
> >Sections are allowed to dry and blocked for 30 min in 10% normal mouse
> serum
> >in PBS (blocking buffer).
> >Sections are stained in blocking buffer, washed and then stained with
> >secondary (also in blocking buffer). We wash in PBS with 0.1% tween.
> >Having done some searches on histonet I see that tween should probably
> not
> >be used. Furthermore I have tried fixing in 3.2 paraformaldehyde with the
> >same result.
> >Sections that are treated as above without antibody look exactly the same
> as
> >sections treated with antibody when viewed under the confocal. Basically
> >lots of auto-fluorescence nicely labelling all the cells.
> >Any help or protocols would be glady appreciated.
> >Scott
> >
> >--
> >Scott Parker, Ph.D.
> >School of Medicine
> >St. Louis University
> >USA
> >_______________________________________________
> >Histonet mailing list
> >
> >
> Gayle Callis
> Research Histopathology Supervisor
> Veterinary Molecular Biology
> Montana State University - Bozeman
> PO Box 173610
> Bozeman MT 59717-3610
> 406 994-6367
> 406 994-4303 (FAX)

Scott Parker, Ph.D.
School of Medicine
St. Louis University
Histonet mailing list

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