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Author Topic: bone IHC  (Read 2396 times)

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Offline ZCherry

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bone IHC
« on: October 18, 2011, 11:55:35 PM »
Hi everyone,
I am doing bone IHC now and have some problems.
The bone tissue which i used for IHC were frozen in -80℃ for about 2 years .I took them out and cut into small pieces and then decalcified for 3 days (till i can cut them by needle) and then do normal fixed and paffirin embedded .The sections were cut into 3mm pieces.The sections were stored in 4℃ .Before IHC, the sections were incubated in 60℃ for an hour.All procedures were done according to protocols DAKO engineer suggested to .But the main problem is after antigen retrieve, the sections is seriously fall out,only some fat tissue left.Then i tried different AR conditions ,such as EDTA 8.0 ,microwave in boiled water for 0,15seconds,30 seconds,1min,5min,15min or high pressure AR for 2 min,only the unantigen retrived and the 15 seconds section have good tissue remained.Others sections almost have no tissue left.But I do the left procedures on every section.On 0 and 15 seconds sections ,we can see some light brown color ,while the PBS negative control sections were blank.The question is whether the brown color are positive ,for the engineer says my tissue will not achieve AR in 15 seconds and would not be positive.Do you have any recommendations for AR on bone tissue?
By the way,  I also do some IHC with other soft tissue with the same antibodies and achieved good result.
Many thanks for all your suggestion and help!

bone IHC
« on: October 18, 2011, 11:55:35 PM »