IHC Related Problem

[Sudheendra Rao N R]

Also..
we always prepare primary in blocking..so after blocking we give a mild wash
with PBS...and then put primary..so that washing step is not to be taken
seriously.

Deepti and Sudheendra.


On 12/18/07, Sudheendra Rao N R <sudhee26 from gmail.com> wrote:
>
> 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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> > >
> >
> >
>
>
> --
> Think before agree
> Think before you nod
> but STOP thinking
> and You Are God
>



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