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Author Topic: P16ink4a  (Read 16041 times)

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Offline jellyjean

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P16ink4a
« on: May 20, 2003, 05:03:52 PM »
Does anyone have a good working protocol for staining with P16ink4a?
My trial runs have not been very successful.

P16ink4a
« on: May 20, 2003, 05:03:52 PM »

Offline richard03

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P16ink4a
« Reply #1 on: May 20, 2003, 07:51:46 PM »
Are you using formalin-fixed, paraffin embedded tissue sections?

Labvision has P16ink4a antibodies that work with FFPE tissue sections based on the pictures showed on their website, and they usually have working protocols in the datasheets.

http://www.labvision.com/ab.cfm?first=AntiBody&second=25

http://www.labvision.com/ab.cfm?first=AntiBody&second=887

Richard

Offline jellyjean

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P16ink4a on cell smears
« Reply #2 on: May 20, 2003, 08:24:16 PM »
The antibody that I am using is from BD (Becton Dickenson) and I am trying to use it on  thin prep smears, but I am first trying it on my control slides which are colon CA biopsy sections.  I tried using 1:100 and 1:125 dilution and got faint staining.  Any tips?  I am currently following protocol from BD and had hard time with antigen retrieval.

Offline ole

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P16ink4a
« Reply #3 on: May 21, 2003, 04:13:31 AM »
Hi
Novocastra have a p16inka4 antibody that work good on FFPET (NCL-p16-432). They recommend citrate buffer pH 6.0, but i find high pH buffer too work better (EDTA pH 8,0 or TE pH9,0).
They have not tested it on frozen sections and neither have we.
Datasheet contains working protocol.

Offline richard03

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P16ink4a
« Reply #4 on: May 21, 2003, 09:57:12 AM »
You may want to try the following:

1) Bring up the concentration to 1:50 or higher.
2) Do overnight incubation at RT instead of 1 hour incubation.
3) Try different antigen retrieval methods since manufacture's recommendation is not always the best one.
4) You may need to use some detergent such as Triton X-100 for the smear samples.

Good luck!

Richard

Offline jellyjean

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Pretreatment for alcohol fixed specimens (cell smears)
« Reply #5 on: May 21, 2003, 12:30:07 PM »
Hi, Richard;
thanks for the info. I incubated my specimens overnight last night.
Hopefully it gives better results.
And I was wondering if there is any pretreatment that I need to be doing to my alcohol fixed smears  that I may be missing in my protocol.What function  does  detergent such as Triton X-100 on the smear samples?
People have told me antigen retrieval need not be done on alcohol fixed specimens.  My specimens were fixed in 95% alcohol for 3days.

And also, You mean try more concentrated antibody?
I tried 1:75, 1:100 and 1:125 and 1:75 did not give any positive staining, but 1;125 gave the strongest even though it was not the strongest I would've liked to see.
Do you recommend using more dilute concentration for the cell smear?
Deadline is near and my project isn't going. :(
Thank you so much for all your help!

Offline richard03

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P16ink4a
« Reply #6 on: May 21, 2003, 02:05:16 PM »
Antigen retrieval is for formalin-fixed, paraffin-embedded tissues. So regarding the alcohol fixed smear samples, there is no need to do antigen retrieval.

The function of Triton X-100 is to break tiny gaps of the cell membrane therefore helping antibody penetration better. However, if the antigen is membrane protein, you better not to use Triton since it will damage the membrane protein. The concentration of Triton x-100 ranges from 0.01% to 0.2% in PBS or blocking buffer. I would suggest to use around 0.05% for your cell smears since they are more fragile.

The concentration of primary antibody is sometimes tricky. I once had a polyclonal antibody that worked in high dilution (1:2000), but didn't work in low dilution (1:500) with high background. So if the antibody you are using is a polyclonal (rabbit) one, that may explain why the higher dilution one (1:125) worked better than the lower dilution one (1:75). If this is the case, you may want to try higher dilution such as 1:200 or 1:500, etc.  

Another comment I had is that the dilution range you are using is to close from each other. Using a wider range (for example, 1:50, 1:100, 1:200, 1:400, 1:1000) may save your time and effort.

Hope this helps and good luck!

Richard

Offline jellyjean

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P16ink4a
« Reply #7 on: May 21, 2003, 03:23:24 PM »
Is that the case for Polyclonal antibody?  Mine is monoclonal mouse antibody.  and I am using biotinylated secondary antibody (goat polyclonal).  for the secondary antibody, I am using 1:100 dilution.  I can use the same concentration secondary antibody on different concentration primary, right?
My overnight incubation did not work either.
I'll look into using that detergent.
Thanx again !

Offline jellyjean

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Triton x-100
« Reply #8 on: May 21, 2003, 03:44:40 PM »
I just went to check out Triton x-100; there are so many different kinds.
Do you recommend anyone in particular? which company? which formula?

Offline richard03

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P16ink4a
« Reply #9 on: May 22, 2003, 02:47:44 PM »
I always use Sigma's and the Cat# is X-100.

Richard

Offline jellyjean

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success at last!
« Reply #10 on: May 22, 2003, 06:38:20 PM »
Hi, Richard:
My stain came out strong on Colon Ca sections with 1:50 dilution.
Now, I am going to have to get staining on my Thin-prep smears.
Thanks again.

Jean

Offline richard03

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P16ink4a
« Reply #11 on: May 22, 2003, 10:26:23 PM »
Very glad to hear that and good luck with your smear staining!

Richard

Offline ikirbis

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P16ink4a
« Reply #12 on: May 23, 2003, 07:25:51 AM »
Dear Jean,
as a positive control for p16 immunostaining we used cytospins prepared from HeLa cell culture (human cervical cancer cell culture). We got positive staining with p16 on Papanicolaou stained cytospins, however MICROWAVE pretreatment is required. It is strange but microwave pretreatment is mandatory even for immunodetection of some antigens on ethanol fixed smears or cytospins! We have a lot of experiences with this.
have a nice day

Offline jellyjean

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How strange
« Reply #13 on: May 23, 2003, 02:57:44 PM »
Hi, thank you so much for your info.
That is why I kept getting no results!!!!
I left out the microwave treatment because it was fixed in alcohol.
Would pressure cooker method work as well?
My microwave pretreatment on formalin fixed specimen has been unsuccessful.
Thank you so very much.
YOu are a life saver!!!

Offline jellyjean

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one more question
« Reply #14 on: May 23, 2003, 03:00:31 PM »
which dilution factor did you use on your ethanol fixed specimens?
I am thinking about 1:50 and 1:75 since 1:50 came out strong on the paraffin embedded tissue seciton.
Any info will save me a lot of work!
Thanks.

one more question
« Reply #14 on: May 23, 2003, 03:00:31 PM »