Re: [Histonet] mouse spleen confocal

From:Gayle Callis


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 

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 Parker, Ph.D.
>School of Medicine
>St. Louis University
>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)

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