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Author Topic: severe background in fetal spleen staining (dako vwf)  (Read 2836 times)

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Offline Yao

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severe background in fetal spleen staining (dako vwf)
« on: May 28, 2010, 09:31:32 AM »
I use dako vWF antibody(1:400) to stain fetal spleen (E18.5) vessel, and have strong background.Similar results from CD31 staining, no matter  both frozen section or paraffin, of coz, very clear signal in some large vessel.
i use heat ag retrieval (sodium citrate, pH6.0, 950C),25min.
block with 5% goat serum for 30min, RT
vWF was diluted in block serum with 0.1% tritonx100,4oC,O/N
wash with PBST, 10min X3
ready to use 2nd ab, rt, 1hr

very negative in control without primary antibody
actually, i am rookie in spleen, i don't know whether this is normal or something need to be optimized. I also used CD31 and got similar result with vWF
 
through EM, i can see more capillary vessels, but it seems that the signal is generally in bigger vessels.

hope someone can give some suggestions.

severe background in fetal spleen staining (dako vwf)
« on: May 28, 2010, 09:31:32 AM »

Offline richard03

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Re: severe background in fetal spleen staining (dako vwf)
« Reply #1 on: May 28, 2010, 04:00:05 PM »
You need to reduce primary concentration greatly, i.e., 1:1000, 1:2000. Give it a try.

Offline gula

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Re: severe background in fetal spleen staining (dako vwf)
« Reply #2 on: May 29, 2010, 04:10:36 AM »
yes, i said negative control is good, thanks all the same
« Last Edit: May 29, 2010, 08:29:19 AM by Yao »

Offline Yao

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Re: severe background in fetal spleen staining (dako vwf)
« Reply #3 on: May 30, 2010, 08:09:54 PM »
You need to reduce primary concentration greatly, i.e., 1:1000, 1:2000. Give it a try.

i tried 1:800, and got better view, thanks a lot

Re: severe background in fetal spleen staining (dako vwf)
« Reply #3 on: May 30, 2010, 08:09:54 PM »