Felix,

Please send me your photo via private email, I will take a look at it 
without disturbing Histonet.   I will be happy to visit with you privately=20
on how to get IHC results also.

Both your primary and secondary antibody concentrations are high.  We do 
immunofluorescent (IFA) staining for CD4 and CD8 using the same antibodies=20
you use and see definite patterns in the spleen (excellent positive control=20
- it could be your liver does not have these cells present.)

We never use Tween 20 for IFA work, only for IHC. Buffers can contain 
normal serum matched to host of secondary, approx 0.2% to keep section from=20
drying out during staining.  We prefer normal serum to BSA and if we had to=20
use BSA, it is protease/immunglobulin free from Jackson.  Most of the time,=20
we keep the buffer pure, without any additives.  After the secondary, we 
rinse 5X with pure buffer to insure there is no fluorophore sitting around=20
- glowing garbage.

Our FS are no thicker than 5 um.  For IFA,  we prefer to section, air dry 
overnight then fix with either cold acetone the next day, air dry 20 min 
and proceed with staining.  Instead of air drying FS, storing, fixing, air=20
drying and storing again - simply air dry frozen sections for a few hours 
at RT, fix in cold acetone, air dry 20 min to get rid of acetone,  then 
store at -80C in a box containing a bag of 16 mesh silica gel to keep FS 
dry.  Bring out acetone fixed FS on day of staining but DO NOT OPEN BOX for=20
30 min to allow these fixed FS to equilibrate to RT.  Water condensation is=20
the enemy, you want DRY FS.    These FS will keep for a few weeks.   Never=20
take a box out of freezer, then pull a few slides for staining, as 
freeze/thaw is very damaging to these antigens.  Use separate 
boxes/staining run or slide mailers in baggie with large tea bag of silica=20
gel so you can pull slides without exposing other sections to freeze/thaw.

You can get rid of some nonspecific binding by using a normal serum block 
of 5% goat serum/1% mouse serum, be sure the serums are heat inactivated, 
cooled and and microfiltered for purity/sterile conditions.  We use this 
NSB before primary application, and for dilution of secondary.

If is a good idea to do a panel dilution of your primary antibodies 
starting with target concentration of 10 ug/ml so you optimize your working=20
concentration.  Secondary antibody is usually used at approx 2 to 5 ug/ml,=20
or usually about 1:250 of a 0.5 mg/ml stock concentration of 
secondary.   It is important to purchase the goat anti rat secondary 
antibody adsorbed to mouse to prevent nonspecific binding.  We  prefer a 
secondary (goat antiRat, adsorbed to mouse) that is an F(ab')2 fragment of=20
IgG to prevent fc portion of IgG molecule from binding to fc receptors on 
mouse tissues although whole IgG molecule secondaries will 
work.   Excellent secondaries come from Jackson ImmunoResearch, 
Biosource/TAGO, Rockland and Southern Biotechnology for murine 
work.  Jackson provides recommended concentration/dilution of their 
secondaries for immunostaining work (IFA/IHC? on their specification sheets.

You can use TRIS buffered saline or Dulbecco PBS  containing 0.2% normal 
serum matched to host of secondary, and do not add Tween 20, not needed for=20
IFA work.

Normal serum block 30 min at RT.

For IFA work, we tend to work with a slightly higher concentration of 
primary as compared to our IHC with the same primaries you work with.

For IFA work, try diluting your CD4 ((0.5mg/ml) 1:200 to 1:250, and dilute=20
in 2% to 5% goat serum.
For negative control the IgG2a must be the same concentration as your CD4 
primary.

For CD8 (0.5mg/ml) , we dilute 1:50 in diluent as CD4  (for CD8 IHC work, 
we dilute 1:100)
IgG2a must be same concentration for CD8 negative control

Incubation is 30 min at RT

Secondary goat antiRat - FITC is diluted 1:250 to 1:300 in 5% goat, spun 
down to get rid of fluorophore protein aggregates before application to 
section and incubated at RT for 30 min IN THE DARK.

Rinse well, coverslip.  We prefer Molecular Probes antiFade, Prolong Gold 
ready to use mounting media to prevent photobleaching of FITC aothough your=20
media is probably adequate.

At 05:25 AM 3/16/2005, you wrote:
>Dear all,
>
>I'm trying to stain CD4 and CD8 T cells in cryosections from mouse spleen.
>However, with not much success so far. The good thing is that I only see
>staining in spleen (and not in liver, which was embedded in the same block
>together with the spleen). However, I don't really get staining in the
>areas where I would expect to see it and I only see small clusters of cells
>instead of big ones. And the real problem is that I get the exact same
>staining with the Isotype control as with the specific anti-CD4 and
>anti-CD8 antibodies. (I tired to post a picture of the staining on
>www.histonet.org, but so far I always get a message back of delivery
>failure, but I will try again...). Somehow it seems that IgG2a antibodies
>unspecifically bind to spleen but not to liver...
>
>I'm using the following antibodies that are supposed to work  in
>immunohistochemistry:
>Rat anti-mouse CD4 IgG2a, Clone RM4-5, BD 550280
>Rat anti-mouse CD8a IgG2a, Clone 53-6.7, BD 550281
>Rat IgG2a Isotype control Clone R35-95, BD 559073
>
>Secondary Antibody was Goat anti-Rat IgG (whole molecule) - FITC (SIGMA
>F-6258)
>
>The protocol that I was using is the following:
>
>Sections: 10um of mouse liver & spleen embedded in OCT medium
>Dry over night at room temperature
>Fix in 100% Aceton 4°C 10', dry 15' and then store at -80°C until use
>
>Take out of freezer and dry 5'
>Rehydrate 20' in PBS
>Block 20' with 3% normal goat serum in buffer (0.5%BSA, 0.1% Tween20 in
>PBS)
>Wash 5' in buffer
>Incubate with 1° Antibody (see above) 1hour (Dilutions: 1:10, 1:25, 1:50,
>Isotype control 1:10 and 1:50)
>Wash 3x 5' with buffer
>Incubate with 2° Antibody (see above) 1 hour (Dilution 1:25)
>Wash 3x 5' with buffer
>Mount in Fluoromount G (Electon Microscopy Sciences)
>
>I also tried to do Immunohistochemistry with the same antibodies using
>Vectastain Elite ABC Kit (Rat IgG) (Vector Laboratories) Peroxidase system
>with NovaRed as substrate and I get a similar result as with the
>fluorescence staining.
>
>I would very much appreciate your advice or to hear your opinion. Maybe
>somebody may recommend another protocol / antibodies that should work on
>cryostat sections.
>
>Thank you very much in advance,
>
>Best Regards,
>
>Felix
>
>-------------
>
>Felix Rintelen (Post Doc Fellow)
>Serono Pharmaceutical Research Institute
>14, Chemin des Aulx
>1228 Plan-les-Ouates
>Geneva, Switzerland
>
>
>
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Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)



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