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Author Topic: Lower temperature for long time vs. high temp for short time  (Read 15629 times)

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Offline ImmunoNYC

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Lower temperature for long time vs. high temp for short time
« on: September 24, 2005, 12:16:52 PM »
I am trying to improve some techniques for antigen retrieval for some tricky mouse bone specimens we have. I get beautiful immunos but sometimes the calcified bone somewhat comes off the slide or rips leading to ugly sections. This only happens when we use our standard antigen retrieval in a veggie steamer. If I use enzymatic or no retrieval the sections are fine.

I am wondering if I can change my methods to a lower temperature water bath for say overnight instead of the 95 deg C for 20 minutes which we routinely do.

Has anyone compared a lower temperature antigen retrieval to a standard one? Will it work? Advice?

Lower temperature for long time vs. high temp for short time
« on: September 24, 2005, 12:16:52 PM »

Offline richard03

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Lower temperature for long time vs. high temp for short time
« Reply #1 on: October 04, 2005, 01:14:53 PM »
Hi MaximinaNYC,

There is a recent post by Dr. Chris van der Loos on Histonet about Low temp Antigen Retrieval for Bone Sections. Here is his answer to the question. Hope this helps.

Quote
Dear Kim and others,

   We  have  been  testing  (not  for  bone  unfortunately) the overnight
   antigen  retrieval  at  60-70C  with  both citrate pH6.0 and Tris-EDTA
   pH9.0  and compared the staining results with the short boiling method
   (15  min  + 20 min cool-down). After overnight antigen retrieval using
   citrate6.0  the staining intensity was much less than with the boiling
   method.  However, after overnight antigen retrieval using Tris-EDTA9.0
   compared  with  the short boiling procedure the staining intensity was
   sometimes  similar,  sometimes less, sometimes even stronger. As ever,
   it varied  per  antigen. Interestingly, during the overnight procedure
   the  "fatty  tissue  parts" stayed much better on the glass than after
   the  short  boiling  procedure.  Nearly the same was described by Junn
   Chavez and Tim Morken in their NSH-poster.

   Overnight  antigen  retrieval  at  60-70C  is  to my opinion something
   worthwhile  to  investigate  further.  It's definitely interesting for
   those  working  with  tissues  that  tends  to fall off during antigen
   retrieval using the short boiling procedure.

   Chris van der Loos, PhD
   Dept. of Pathology
   Academic Medical Center M2-230
   Meibergdreef 9
   NL-1105 AZ Amsterdam
   The Netherlands

   phone:  +31 20 5665631
   fax:    +31 20 6960389



   Date: Mon, 26 Sep 2005 11:34:06 -0700 (PDT)
   From: Kim Merriam
   Subject: [Histonet] Low temp Antigen Retrieval for Bone Sections
   To: Histonet
   Hello All,
   I  will  be  performing  some  IHC  on  mouse  femurs  (about 12 or so
   different  antibodies).   In  the  past,  I  have had a lot of trouble
   keeping  these  sections  on  the  slides  during HIER.  I did a quick
   search on the archives and there were lots of suggestions, but nothing
   definitive.
   I  was reading an article about "low temp AR", overnight at 60C in TEG
   buffer,  pH  9.0.  Has anyone tried this method?  Also - what does TEG
   buffer stand for?
   Any help would be greatly appreciated.
   Kim
   Kim Merriam
   Novartis
   Cambridge, MA

Offline ole

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Lower temperature for long time vs. high temp for short time
« Reply #2 on: October 04, 2005, 02:20:32 PM »
Hi
60deg/C - 24h, 95deg/C - 40mins, 100deg/C - 15-20mins is common, temp vs times. I have thought about trying 80deg/C on bonemarrow for a while but ... hmm maybe next year.

I think lower temp is better - "prevent" bone from falling off, and believe i have read somewhere 80deg/C beeing better than 60deg/C reg retrieving of  several antigens.

Well thats my vague reply. cant remember the time at 80deg/C :oops:

I will try to find the paper/article where its from the 80deg/C temp on bone sections.

Ole

Offline ImmunoNYC

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Lower temperature for long time vs. high temp for short time
« Reply #3 on: October 04, 2005, 11:47:18 PM »
Thanks. I would really appreciate that, I may try 80 deg C soon too.

Quote from: "ole"
Hi
60deg/C - 24h, 95deg/C - 40mins, 100deg/C - 15-20mins is common, temp vs times. I have thought about trying 80deg/C on bonemarrow for a while but ... hmm maybe next year.

I think lower temp is better - "prevent" bone from falling off, and believe i have read somewhere 80deg/C beeing better than 60deg/C reg retrieving of  several antigens.

Well thats my vague reply. cant remember the time at 80deg/C :oops:

I will try to find the paper/article where its from the 80deg/C temp on bone sections.

Ole

Offline ImmunoNYC

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Lower temperature for long time vs. high temp for short time
« Reply #4 on: October 04, 2005, 11:48:41 PM »
Thank Richard, I had seen this on Histonet and thought it was extremely informative. Thanks for the reminder!

Quote from: "richard03"
Hi MaximinaNYC,

There is a recent post by Dr. Chris van der Loos on Histonet about Low temp Antigen Retrieval for Bone Sections. Here is his answer to the question. Hope this helps.

Offline ole

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Lower temperature for long time vs. high temp for short time
« Reply #5 on: October 10, 2005, 09:48:06 AM »
hi
Couldnt fint the paper/article i was looking for.
60deg/C is better reg preservation of collagenousfibres.
i think 80deg/C retrieval is about 12-16h (over night)
I think theres some info in this one below, regarding the issue?

Koopal SA.....................
Low temperature heating overnight..................
Applied immunohistochemistry  6:228   (1998)

Ole

Lower temperature for long time vs. high temp for short time
« Reply #5 on: October 10, 2005, 09:48:06 AM »