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Cryosection problem


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#1 Rnotk

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Posted 14 June 2009 - 02:48 PM

Hi,
I am doing IHC and H&E staining of cryosection, and having problem of tissue coming off from slide while I am staining.

What I normally do to prepare slide is

cut section (12um),
put on slide
briefly melt the tissue on slide with temparature from my finger
then store at -80 until staining

when I stain, I also heat the slide at 60 C for 30min to dry the tissue.

I looked up some protocols on line and
I will try to re-fix slide with 4% PFA/0.25% glutaldehyde solution

But Do you guys have any other suggestions about this issue???
Any suggestion help
FYI my sample is already fixed before cryo-preserved and some of the sample is very tiny (piece of nerve)

Thank you in advance

#2 bachai

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Posted 14 June 2009 - 05:59 PM

Hi,
I am doing IHC and H&E staining of cryosection, and having problem of tissue coming off from slide while I am staining.

What I normally do to prepare slide is

cut section (12um),
put on slide
briefly melt the tissue on slide with temparature from my finger
then store at -80 until staining

when I stain, I also heat the slide at 60 C for 30min to dry the tissue.

I looked up some protocols on line and
I will try to re-fix slide with 4% PFA/0.25% glutaldehyde solution

But Do you guys have any other suggestions about this issue???
Any suggestion help
FYI my sample is already fixed before cryo-preserved and some of the sample is very tiny (piece of nerve)

Thank you in advance


Frozen tissue sections will keep coming off your slides unless you precoat slides with something e.g. HistoGrip or APES. Search the Internet to chose a supplier and get methods how to do it. It is quite boring but essential step to assure your success with both paraffin (IHC following antigen retrieval) and frozen tissues (both IHC and H&E). Good luck!

#3 Dominic

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Posted 15 June 2009 - 05:40 AM

i use apes on all histo or cyto slides
wax sections are floated on in a water bath and baked on overnight (37C)(some primaries dont like higher heat)

frozen sections i press onto the slide using the cryostats plate

the rest is down to your technique as an immunostainer - always drip at the same pount on slide, not directly on the tissue, dont let the tissue dry out.

btw - the pfa/glut will fix your tissue - which means more ER - not what you want really - as ER is main reason for removal of tissue

dom




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