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Author Topic: Rat anti-PECAM  (Read 6892 times)

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Offline madeleine huey

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Rat anti-PECAM
« on: March 10, 2006, 10:07:05 PM »
Hello Richard,
I have a question regarding your Proteinase K digestion. How did you prepare the digestion & incubation?  Did you put the slides inside a large volume of 200 ml or 50 ml Proteinase K inside the Tessue-Tek/Coplin Jar, or did you applied suffient Prot. K (ie. 100 ul - 200 ul P.K./slide) on top each slides, then incubate in the 60ēC incubator?  
I think there's a crucial information I am missing here, because I have try your protocol 5 times, and none of them work.  The slides are either over-digested (lost nucleus) or under-digested (perfect morphology, but no positive stain).  Three out of 5 experiments I used 40 ml Prot. K in 50 ml Coplin Jar, and 2 experiments with 20 ml Prot.K in slide mailer.  All Prot. K are pre-warmed in 60ēC water-bath first, then incubation timing start count down at this point.  Following are the time frame I have done.  Total 5 slides  @ 60ēC for 10', 15', 20', 25', & 30' & cool @ RT for 10 min.  For 4 slides with 20 min @ 60ēC, cool @ RT for 10', 15', 20', & 25'.  Never the less, when I used TV= 40 ml Prot. K, they are over-digested, but when I switch to 20 ml Prot. K in slide mailer, then they are under-digested with Negative staining.  
Your advice and suggestion will be greatly appreciated!
M

Rat anti-PECAM
« on: March 10, 2006, 10:07:05 PM »

Offline richard03

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Rat anti-PECAM
« Reply #1 on: March 11, 2006, 06:32:43 PM »
Hi M,

As matter of fact, I have recently done some mouse tumor sections using PECAM-1, and it did not work at all. But the positive control of mouse skin worked well.

So I started to think that the PECAM-1 may only work on selective endothelial cells. In another word, it may not bind to certain type of endothelial cells.

However, Zinc fixed tissues almost work 100% of time. So I started to recommend to use Zinc fixative for CD31 on mouse tissues to save a lot of time trying the method with inconsistant results.

Anyway, I use capillary gap (MicroProbe On) slides for most of my IHC work. For PK pretreatment, I put two slides face to face, and place it to a staining tray filled with PK solution. Wrap with parafilm, and place it in 60 C oven for 20-30 minutes. Then let it cool at RT for 20 minutes.

Hope this helps and good luck.

Richard

Offline ImmunoNYC

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Re: Rat anti-PECAM
« Reply #2 on: March 13, 2006, 12:43:34 AM »
At 60 deg C won't Proteinase K be deactivated as it is a protein?????????? Why are you doing it at 60 deg C? It is standard to do proteinase K at 37 deg C and this is what I do with no problems.[/color]

Quote from: "madeleine huey"
All Prot. K are pre-warmed in 60ēC water-bath first, then incubation timing start count down at this point.  Following are the time frame I have done.  Total 5 slides  @ 60ēC for 10', 15', 20', 25', & 30' & cool @ RT for 10 min.  For 4 slides with 20 min @ 60ēC, cool @ RT for 10', 15', 20', & 25'.

Offline madeleine huey

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Rat anti-PECAM
« Reply #3 on: March 15, 2006, 04:07:04 PM »
Hello Richard,
Thank-you for your info. and suggestions.  
I have another question relating to this rat-PECAM (13.3) ab.  Have you ever used 4%PFA as a fixative and compare with the Zinc fixative?  Do 4%PFA paraffin section work on this ab?
Thank-you for your suggestion again!
M

Offline richard03

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Rat anti-PECAM
« Reply #4 on: March 15, 2006, 08:05:08 PM »
I have never compared this antibody side by side for Zinc fixative, 4%PFA, and Formalin. However, I have done with these fixatives separately. My impression is that the Zinc fixative worked the best (100% of times), and 4% PFA worked (sometimes it did not work), but formalin was the worst (only worked for skin).

If you already have tissue fixed with 4%PFA, give it a try. Otherwise I would definitely go with Zinc fixative.

Richard

Quote from: "madeleine huey"
Hello Richard,
Thank-you for your info. and suggestions.  
I have another question relating to this rat-PECAM (13.3) ab.  Have you ever used 4%PFA as a fixative and compare with the Zinc fixative?  Do 4%PFA paraffin section work on this ab?
Thank-you for your suggestion again!
M

Rat anti-PECAM
« Reply #4 on: March 15, 2006, 08:05:08 PM »