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Author Topic: urgent help for p-AKt kit from CST(catalog 8100)  (Read 6972 times)

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Offline Prashant

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urgent help for p-AKt kit from CST(catalog 8100)
« on: March 28, 2006, 02:04:46 AM »
Hello, I am using cell signaling technology p-Akt IHC kit(catalog number8100). The kit uses rabbit primary antibody against p-AKt Ser 473,biotinylated secondary antibody with avidin DH & HRP & Nova Red as a substrate.
My problem:1) Using the kit Non specific nuclear staining of strong intensity in normal tissues . This staining persists to a mild degree in negative control slides(where I omit primary antibody). AS a counter stain we are dipping the slides in Gills hematoxylin for 2 dips(should we increase this)
2) I am unable to use their primary pre diluted antibody with DAKO envison kit so as to get final staining with DAB.I tried using all the kit components till the nova red substrate chromogen step where I substituted DAB but it did not work. Then I tried using their pre diluted antibody with DAKO envison kit  :!:  :?:
I need urgent help on this. Anyone got any suggestions. If anyone has used the CST kit on FFPE sections their experience will be invaluable.[/b]

urgent help for p-AKt kit from CST(catalog 8100)
« on: March 28, 2006, 02:04:46 AM »

Offline excalibur

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urgent help for p-AKt kit from CST(catalog 8100)
« Reply #1 on: March 28, 2006, 11:15:46 AM »
Are you blocking endogenous peroxidase with H2O2?

Are you blocking for endogenous avidin and biotin?
Paula Keene Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
5830 N Blue Lake Dr.
Norman, OK 73069
405-759-3959
www.excaliburpathology.com

Offline Prashant

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urgent help for p-AKt kit from CST(catalog 8100)
« Reply #2 on: March 28, 2006, 12:36:36 PM »
thanks!
when I am using the kit i follow their instructions & use all the  reagents supplied with the kit. I do not use any additional reagents or blocking solutions.
While trying to make their pre diluted primary antibody from kit stain with DAB I did block peroxidase with H2O2. But I did not use any avidin & biotin specifically. I just used Bovine albumin serum.

Offline excalibur

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urgent help for p-AKt kit from CST(catalog 8100)
« Reply #3 on: March 28, 2006, 01:35:09 PM »
Alrighty, most kits do not come with the blocking reagents. But they do include a list of additional reagents needed.

With ALL HRP detection systems you will need to block endogenous peroxidase with H2O2 EVERYTIME no matter what chromagen.

If you still have high background you will also need to block avidin and biotin. 5% skim milk and egg white in PBS can be used for this and you will not spend hundreds on commercial solutions.

;)
Paula Keene Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
5830 N Blue Lake Dr.
Norman, OK 73069
405-759-3959
www.excaliburpathology.com

urgent help for p-AKt kit from CST(catalog 8100)
« Reply #3 on: March 28, 2006, 01:35:09 PM »