How to eliminate the background staining in the dentin slice - (Nov/09/2007 )

I want to stain the cell on the dentin slice(bone slice).I put the slice in the 24 wells plate.I can get good staining in the cells on the plates,just besides the slice.but the cells on the slice are hard to discern,because the background staining in the slice is too heavy.I rinsed the slice in pbs for 5 times,5mins per time,in the swing bed,but that seemed unuseful.Since the cells besides the slice were stained well,the cells on the slice should also be stained.I think maybe the dentine slice absorb the fluorescent and cannot be washed out.Can anyone give me some advices?Thanks in advance.

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you are going to have to be much more specific than that
stain? antibodies or general fluor stain
do you block?
etc etc

dom

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yes,i have used 1%BSA to block the unspecific staining(37deg,30mins).The fluorecence inside the cells(on the glass,just besides the dentines slice) was very good.there was no unspecific staining.
i also stained the nuclei with DAPI.sill the background was too heavy to discern the cells.
so i think it was not because of the unspecific staining.maybe the fluorecence substance permeated into the dentine slice cannot be washed away.
thanks for your reply.
any more suggestion?

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stain? antibodies or general fluor stain

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1% bsa may be too weak. try higher (we use up to 5% to block other procedures).

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I used 5%bsa before.
I want to stain the actin with phalloidin and the technician of the invitrogen suggest 1%bsa and said it is the best concentration for phalloidin.
I try 5% bsa again.Hope it works.

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you could try a range of concentrations and use that which gives you the best overall result. if invitrogen recommends 1% then you may find that 2% works better, overall, than 5%.

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