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Author Topic: ki 67  (Read 7717 times)

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Offline akleinschmit

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ki 67
« on: January 30, 2006, 01:30:25 AM »
I see that it is recommended not to use PIER for ki 67, but I am really trying to avoid HIER for my assay.  What would you recommend for the best conditions for ki 67 using proteinase k in mouse large intestine sections?

ki 67
« on: January 30, 2006, 01:30:25 AM »

Offline excalibur

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ki 67
« Reply #1 on: January 30, 2006, 12:12:21 PM »
Sorry, you can't avoid HIER forever. I suggest you purchase a rice steamer instead of microwaving.
Paula Keene Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
5830 N Blue Lake Dr.
Norman, OK 73069
405-759-3959
www.excaliburpathology.com

Offline akleinschmit

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ki 67
« Reply #2 on: January 30, 2006, 02:40:31 PM »
I would like to laser cut my Ki-67 cells post IHC.  So I need a antigen retrieval technique that doesn't use heat and permanently complex the tissue to the glass slides:( .  Thus any suggestions for protease epitope retrieval would be greatly appreciated for mouse monoclonal Ki-67 :D

Offline excalibur

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ki 67
« Reply #3 on: January 30, 2006, 03:12:15 PM »
The sections should be placed on a slide with some form of adhesive, (silane, poly L lysine, gelatin) and then heated to dry any water from the waterbath before deparaffinization or the sections will wash off the slide during staining.

Unless you doing free floating staining.

One alternative is to cut fresh, frozen sections so no antigen retrieval is needed. But you still need an adhesive slide or the section will wash off.
Paula Keene Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
5830 N Blue Lake Dr.
Norman, OK 73069
405-759-3959
www.excaliburpathology.com

Offline ImmunoNYC

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ki 67
« Reply #4 on: January 30, 2006, 11:14:41 PM »
Can you *not* do LCM after HIER? I didn't know that ..... it's funny that you say you can't do it as it complexes the tissue to the glass b/c practically I find it does the opposite, sections love to come off after HIER not stick on!!![/color]

Offline akleinschmit

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ki 67
« Reply #5 on: February 02, 2006, 09:58:48 PM »
When performing IHC for the downstream use of LCM, a special film for the LCM technique is applied to a blank slide which tissue is placed on.  During IHC on the tissue, liquid often gets trapped between the film layer and the slide during epitope retrieval using the HIER method.  Thus, I would like to find a alternative method of epitope retrieval for Ki-67.  Any suggestions would be appreciated :)

Offline ImmunoNYC

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ki 67
« Reply #6 on: February 02, 2006, 10:58:44 PM »
Are you doing paraffin sections? If so, I just suggest you try different types of enzymatic retrieval on a section of spleen or tonsil and run them alongside the same tissue done with HIER and see what gives you comparable results.

Try pepsin and proteinase K for varying times and compare RT to 37deg C. With about 20 slides you should be able to get some good answers.

Sorry I can't be of more help.
[/color]

ki 67
« Reply #6 on: February 02, 2006, 10:58:44 PM »