IHC Related Problem

[Sudheendra Rao N R]

Hi POW, Susanne

Thanks for the suggestions.

1. we have tried Alexa 594 conjugated sec 1:1000
2. Here we are not using primary at all..only sec gives this staining..
3. we however wish to try this combination (primary 1:250, sec 1:2000)
4. we have done perfusion of rat prior to fixation with pbs.sections were
directly put in DAB without quenching..but not significant staining was seen
(?no or low endogenous peroxidase. also our staining was seen more in neuron
like cells and not blood vessels-which indicates good perfusion)
5. we are not using formalin fixed tissues..we use paraformaldehyde to fix
the animal tissue after pbs perfusion.
6. we have also tried different dilutions of biotinylated secondary.
7. we have also tried rabbit polyclonal antiGFAP..and in its -ve control
stain (i.e without primary) no staining was seen as seen in antiMouse
secondary earlier..that makes it specific to antiMouse.
8. the protein atlas is very nice..but regional pattern cant be seen..i
guess they used punched tissue for staining.
9. we will send the pic if needed.

still in dire need of help.

Deepti and Sudheendra.



On 12/18/07, Pow Joshi <pow.joshi from gmail.com> wrote:
>
>
>
>  On 17/12/2007, Sudheendra Rao N R <sudhee26 from gmail.com> wrote:
> >
> > Hello all,
> > We are trying to do IHC on free floating 30 microns rat brain sections.
> > Primary is antiGFAP (Mouse Monoclonal-1:1000) and secondary is antiMouse
> > (Horse: 1:250), by ABC-DAB method.
> > But we are getting reproducible nonspecific staining in SPECIFIC regions
> > of
> > the rat brain
> >
> > Our IHC method is
> > 1. TBS / PBS Washing
> > 2. Quenching for 10 min
> > 3. TBS/ PBS washing
> > 4. Blocking with 3%-5% Normal Horse Serum (also tried BSA)
> > 5. Washing
> > 6. Primary
> > 7. Washing
> > 8. Secondary
> > 9. Washing
> > 10. ABC
> > 11. Washing
> > 12. DAB
> >
> > We did a negative control by skipping the primary..but same results..the
> > cells seem to be of pyramidal / immature neuron morphology.
> > We also skipped both primary and secondary, going ahead with ABC-DAB but
> > no
> > staining (rules out endogenous biotin).
> >
> > We got convinced that only secondary can give nonspecific staining..we
> > tried
> > following methods-(Quenching-Blocking-Secondary)
> >                  1. increasing dilutions: from 1:250 to 1:1000 and then
> > ABC-DAB..still we are getting the same nonspecific staining..
> >                  2. Texas Red conjugated antiMouse (Horse) as secondary
> > and
> > got the similar pattern (no ABC method)
> >                  3. used antiRabbit(Goat) without primary..no staining
> > (looks like problem is specific to antiMouse)
> >                  4. Reagents were changed, new vials (lots) were used,
> > crucibles were changed..6-7 different brain sections were used..same
> > result
> >                  5. This post-secondary antibody stain looks
> > predominantly
> > cytoplasmic, also present in axons
> >                  6. regions where staining is seen : somato sensory
> > cortex,
> > hippocampus, inferior temporal lobe, SVZ etc
> >                  7. Increasing blocking serum (Horse/Goat): 3 to
> > 5%--same
> > results
> >                  8. We have also tried NovaRed--same results.
> >
> > So, this antimouse antibody is binding to some proteins expressed in
> > normal
> > rat brain..??
>
>
>
>
> I don't know too much about DAB staining ... however I have used the
> secondary at 1:1000 dilution for alexa 488 or 594 conjugated with fairly
> specific staining....perhaps the endogenous peroxidase was'nt properly
> "quenched" ... check if the hydrogen peroxide is really good (If you have'nt
> already done so). I would also suggest using the priomary at higher
> concentration of 1:250 and lower secondary rather than the other way around
> ..... ca'nt think of anything else for the moment.
>
> best,
> Pow
>
>
> in dire need of help.
> >
> > regards
> > Deepti and Sudheendra
> > NBRC, Gurgaon, India
> >
> >
> > --
> > Think before agree
> > Think before you nod
> > but STOP thinking
> > and You Are God
> > _______________________________________________
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> > Methods from net.bio.net
> > http://www.bio.net/biomail/listinfo/methods
> >
>
>


-- 
Think before agree
Think before you nod
but STOP thinking
and You Are God