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Methyl Binding Protein-1 (MBD1) / Immunofluorescence


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#1 SelcenC

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Posted 05 October 2011 - 09:07 PM

Hi,

I am currrently on anti-MBD1 staining optimisation in mouse embryonic fibroblasts for immunofluorescence.
I increased blocking serum concentration. And reduced primary antibody concentrations. But non-specific IgG is highly seen so far (with FITC). I fixed cells 4%PFA for 30min, then permeabilise PBS with tween-20 and triton-x.

I am wondering if anyone does specifically work on MBD1 staining for immunostaining...

Thanks and good luck to everyone !

Selcen

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#2 methylnick

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Posted 11 October 2011 - 02:29 AM

Do you have a representative image? I have done MBD2 long long ago and from memory it stains the chromosomes quite evenly with centrmere as more intense.

#3 SelcenC

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Posted 26 October 2011 - 06:37 PM

Hi,

I just want to let you those interested know.

Optimisation is complete for MBD1 immunostaining in mouse embryonic fibroblasts.

The protocol is as follows:

1. fix cells with 4%PFA for 30min at room temp
2. wash cells with 1XPBS (with 0.05% tween-20) for 10min X 3
3. permeabilise with PBS (including 0.5% tween-20 and 0.5% triton-x) for 40 min. at room temp
4. 4N HCl treatment (including 0.1% Triton-X)for 10 min at room temp
5. wash cells with 1XPBS (with 0.05% tween-20) for 10min X 3
6. Block cells with 30% GOAT serum at 4C overnight
7. Primary antibodies in 2%BSA/PBT (5% goat serum included in final volume) as 1:50 at 4C overnight (Important: USE rabbit IgG stock as 0.2 mg/ml)
8. wash cells with 1XPBS (with 0.05% tween-20) for 10min X 3
9. Secondary antibody incubation with FITC (1:200)in 2%BSA/PBT for 1 hour at room temp (DARK)
10. wash cells with 1XPBS (with 0.05% tween-20) for 10min X 3
11. last wash with 1xPBS
12. Mount slides

The products included in this protocol:

Rabbit IgG control: Sigma I5006
MBD1 antibody: Abcam ab3753
FITC: Sigma F1262 (anti-rabbit in goat)

Enjoy !!

:)

- Selcen -





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