Re: [Histonet] Bone adhesion problems after HIER

From:"Andrea Hooper"

Hi Rene,

I think the IHC problems only arose b/c Patrick 
dropped the temp of retrieval down to 70 deg C. I 
can confirm from personal experience that for 
most antigens it doesn't work. Even 80 deg C (the 
point at which the sections start to come off 
Plus slides) is largely ineffective. 95-99 deg C 
is critical retrieval temp in my experience.

I am going to try those slides recommended by 
Nancy, maybe Patrick can do the same (or your 
Elmer's method which for me isn't an option as we 
don't cut our own sections anymore so cannot 
control those parameters too easily).


>   I am somewhat confused about your e-mail, 
>because it is titled "bone adhesion problems" 
>and at the end it seems that you solved that 
>problem, but are having problems with the IHC 
>   1-for the adhesion: I always used 1 mL of 
>Elmer's glue in a regular 2L water bath and 
>"fished" the sections with superfrost + slides 
>(for both nails and bones). Did not lose 
>sections during HIER.
>   2-about the failure with IHC I cannot try to 
>help you if I do not know what epitope your are 
>looking for.
>   René J.

>"Leung, Patrick"  wrote:
>   Hi,
>I am having problems with cortical bone detaching from the slide during
>the antigen retrieval step. The samples I am staining are paraffin
>embedded mouse tibia.
>I am currently using Superfrost+ slides. The AR is with citrate buffer
>(pH6.1) from Dako performed in a waterbath for 15 minutes. The slides
>are baked overnight at 59C prior to deparaffinizing.
>I have tried using a steamer and microwave, however I still ended up
>with the same results. In addition Superfrost GOLD slides did not help
>in keeping the bone on. I found that performing HIER at 70C for three
>hrs as suggested in the archives to be ineffective in exposing my
>antigen (although the bone did stay attached).
>Do you have any suggestions?


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