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Author Topic: Concentration of Proteinase K to use in paraffin embedd  (Read 12078 times)

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Offline kzejnull@bowdoin.edu

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Concentration of Proteinase K to use in paraffin embedd
« on: March 26, 2005, 04:07:19 PM »
Hi all,
I am an undergrad and for the most part of this semester I've been trying to get some antibodies to work in chicken stomach/gizzard epithelium.  Although I've had some success with DAB method using proteinase K for antigen retrival, I think I could modify the protocol a bit more to get better quality staining. I think that I've been using a too high concentration of proteinase K (10mg/ml for 10 minutes) because the tissue get so damaged and full of cellular debris after the proteinase K treatment that I can barly take good pictures.
What is the concentration of Proteinase K people generally use and for how long?

K

Concentration of Proteinase K to use in paraffin embedd
« on: March 26, 2005, 04:07:19 PM »

Offline richard03

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Concentration of Proteinase K to use in paraffin embedd
« Reply #1 on: March 26, 2005, 08:28:17 PM »
I was not surprised when you said the tissue was damaged so hard since you were using about 500x more proteinase K concentration than it supposed to be. The commonly used proteinase K concentration for antigen retrieval ranges from 10-20 ug/ml. The following protocol that I have been using for years with very good success.

http://www.ihcworld.com/_protocols/epitope_retrieval/proteinase-k.htm

Good luck.

Richard

Offline kzejnull@bowdoin.edu

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Concentration of Proteinase K to use in paraffin embedd
« Reply #2 on: March 27, 2005, 04:27:24 PM »
Hi Richard,

Thanks for the message. I tried a lower conc. of proteinase K and the staining worked beautifully. Thanks for the reply.

K

Offline kzejnull@bowdoin.edu

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Concentration of Proteinase K to use in paraffin embedd
« Reply #3 on: March 30, 2005, 04:57:59 PM »
I have another question. The protocol for general PIER states that after taking slides out of the 37 C water bath they should be allowed to cool at RT for 10 minutes. Should I leave the slides in the proteinase K solution, change the solution with washing buffer or just air dry them.

Thanks so much

K

Offline richard03

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Concentration of Proteinase K to use in paraffin embedd
« Reply #4 on: March 30, 2005, 08:53:21 PM »
When cooling, you should leave slides in pk for 10 minutes and then rinse in PBS or other buffer.

Richard

Concentration of Proteinase K to use in paraffin embedd
« Reply #4 on: March 30, 2005, 08:53:21 PM »