Best instrument for heat retreaval in FFPE

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ssivajothi
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Best instrument for heat retreaval in FFPE

Post by ssivajothi » Thu Feb 01, 2018 7:19 pm

Moved from another thread by moderator:

Which instrument do you use to heat the slides to 95C during HIER on FPE tissues? Is it just a water bath or something different?

Thank you!
Last edited by votti on Sun Feb 04, 2018 4:27 pm, edited 1 time in total.
Reason: Moved off-topic comment to a more relevant section
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votti
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Re: Best instrument for heat retreaval in FFPE

Post by votti » Tue Feb 06, 2018 8:59 am

Hi there,

In the Bodenmiller lab we use a pressure cooker for doing the FFPE heat retrieval.
In the IMC conference in summer I heard also stories of people finding expensive microwaves being the best way to do heat retrieval.
drimm
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Re: Best instrument for heat retreaval in FFPE

Post by drimm » Tue Feb 06, 2018 1:47 pm

We have spent a lot of time testing different antigen retrieval methods, including proteases, pH, pressure and subsequently >100 degree temperatures, and microwaves. Although some of the more extreme conditions can make a difference, they are often not reproducible. We have had reproducibility issues with microwaves, proteases (only use that as an absolute last resort) and pressure cookers. The Rimm lab tries to find antibodies that work in the conditions conducive to use in a CLIA lab. That means 97 degrees without increased pressure. This is usually the max that can be generated on most standard autostaining platforms in CLIA labs.
douglas.linn
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Re: Best instrument for heat retreaval in FFPE

Post by douglas.linn » Fri Feb 09, 2018 8:36 pm

We use the Biogenex EZ Retriever system which Jared Burks highly recommended at the users meeting
http://store.biogenex.com/us/ez-retriever
It is expensive, but maintains a very consistent temperature without risk of buffer boiling over like a standard microwave.
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Jkburks
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Re: Best instrument for heat retreaval in FFPE

Post by Jkburks » Thu Feb 22, 2018 3:05 pm

Looks like I am late to this comment, but we have had dozens of researchers use the Biogenex microwave in Opal labeling for the Vectra where multiple retrievals occur. Also we have had at least a half of dozen researchers use it for the IMC and all report better labeling, including us during our early access testing, which we shared with Fluidigm. These groups have tested multiple modalities and all settle on this microwave as it appears to give the most consistent results for them. As David mentioned it uses constant heat without added pressure, which I will echo I believe is the problem with the pressure cooker approaches and the most optimal option. I will also address his other points on reproducibility as this microwave seems to be easily damaged in shipping (don't know if all microwaves have similar issues or not as that isn't as critical when heating your food). I know the Biogenex system went from shipping in a cardboard box to a wooden crated box on a wooden pallet. If the Biogenex doesn't hold within 3 degrees of target temperature for the duration of the retrieval time it is broken as that is the way it is designed to function. It uses a temperature probe extending from the top of the microwave for constant feedback and power modulation of the microwave to stabilize the temperature. Locally about two dozen labs have purchased these microwaves and I personally know of 4 or 5 that were not functioning upon arrival, which is very unfortunate. However, they were all replaced with functioning units from Biogenex in a timely fashion.

As always, testing for your samples is the best approach.

Jared
ssivajothi
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Re: Best instrument for heat retreaval in FFPE

Post by ssivajothi » Thu Feb 22, 2018 3:18 pm

Thank you for the detailed information. It appears the Biogenex microwave seems like an ideal solution. Can you guys share what the total price of the instrument and any associated/required accessories is?

Also, as you know a unique issue for IMC is finding not only the antibodies which work on FFPE, but making sure the ~30 antibodies you are multiplexing work under the same retrieval condition. Can you share what temperature and time combination have you found to be the best for most antigens? This information would be very useful.

Best,
Santhosh
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Jkburks
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Re: Best instrument for heat retreaval in FFPE

Post by Jkburks » Thu Feb 22, 2018 3:23 pm

The prices for this microwave are all over the place. I have had new investigators claim little budget dollars and get it as low as $2,000. The list price is $6000. It isn't cheap, which is the one big negative. Sorry I normally include that info in every statement I make about the microwave. We found it incidentally as a path lab had it and for reasons we didn't initially figure out they were more successful with Opal (tyramide based) labeling than any one else.

Jared
ssivajothi
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Re: Best instrument for heat retreaval in FFPE

Post by ssivajothi » Fri Mar 02, 2018 3:09 pm

Hi all,

Thank you for the suggestions and sharing your experience with different instruments.

I think I seem to have found a cheaper solution than the special microwave: A SOUS VIDE COOKER!

Yes, it is the same one you use to precision cook steaks and other items. It is essentially a water circulator which can maintain temperature at a 0.1C precision, which is perfect for temperature controlled applications.

I clipped the circulator on to a vessel filled with some water and just set the temperature to the max 99C. I then placed a container with the retrieval buffer in the bath and I was able to achieve stable 95C in the buffer, so its perfect. It takes around 20 minutes for the bath to come to temperature but thats just a minor inconvenience.

The Anova sous vide precision cooler I used for this can be bought for just $100! I think it can be a cheap and effective solution.

Best,
Santhosh Sivajothi
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