Author Topic: Dealing with pre-stained slides  (Read 5873 times)

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Offline MFeirer

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Dealing with pre-stained slides
« on: October 25, 2011, 06:38:48 PM »
Hi,

I'm hoping someone can provide any hints or insights into my problem.  I'm trying to develop a protocol for ICC on fine needle aspirates/impression smears of canine lymph nodes.  I've gotten a handful of antibodies to work on previously unstained aspirates/imprints by using acetone fixation followed by TritonX100 treatment (0.1% in PBS).  I'm now trying to apply this to samples previously Wright-Giemsa stained (methanolic) by applying the above acetone fixation and TritonX treatment once the previously stained slides are dry.  My problem is that only 50% of my antibodies work in this new format, and I'm honestly clue-less as how to proceed.

The two antibodies in question do not work with either methanol fixation alone or with an acetone:methanol mix (50/50).  Has anyone had issues like this before, in terms of ICC on previously stained slides?  Is there any hope for these markers after the original stain based methanol exposure?  Could I increase my acetone fixation time (currently 10 minutes)?  How about my TritonX concentration (0.1%) or time (10 minutes)?

Thanks in advance for any help that can be provided!  Below, if interested, you'll find an outline of my current working protocol.
----------------
1.  Fix in 100% acetone @ room temp (RT) - 10 min
2.  Dry @ RT - 1 hour
3.  0.1% TritonX100 in PBS - 10 min
4.  PBS Wash
5.  Protein Block - Thermo SuperBlock T20 (PBS) - 30 min (Do not rinse, gently blot off)
6.  Primary Ab - O/N 4C
7.  PBS Wash
8.  3% H2O2 - 15 min @ RT
9.  PBS Wash
10.  Secondary Ab - 30 min @ RT (HRP conjugate)
11.  PBS Wash
12.  DAB Substrate - 10 min (Vector Labs)
13.  H20 Wash
14.  Hematoxylin Counterstain - 2 min
14.  H20 Wash

Dealing with pre-stained slides
« on: October 25, 2011, 06:38:48 PM »

Offline MT Scientist

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Re: Dealing with pre-stained slides
« Reply #1 on: October 26, 2011, 11:08:19 AM »
While this isn't a great answer, I remember reading somewhere that MeOH isn't nice to some antigens.  That may be what is going on....

You may need to try a matrix to determine the optimal conditions.

Offline CanuckPhD

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Re: Dealing with pre-stained slides
« Reply #2 on: October 27, 2011, 01:02:08 AM »
You might be able to perform an antigen retrieval on the previously stained sections to overcome the issues with methanol. This might affect the Wright staining but I have no experience in this area. The only way to find out would be to test it.

Of course the optimum plan would be to collect new samples but I am assuming that this would be difficult.

Offline Cardio

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Re: Dealing with pre-stained slides
« Reply #3 on: October 27, 2011, 03:58:01 PM »
Hi,

I'm hoping someone can provide any hints or insights into my problem.  I'm trying to develop a protocol for ICC on fine needle aspirates/impression smears of canine lymph nodes.  I've gotten a handful of antibodies to work on previously unstained aspirates/imprints by using acetone fixation followed by TritonX100 treatment (0.1% in PBS).  I'm now trying to apply this to samples previously Wright-Giemsa stained (methanolic) by applying the above acetone fixation and TritonX treatment once the previously stained slides are dry.  My problem is that only 50% of my antibodies work in this new format, and I'm honestly clue-less as how to proceed.

The two antibodies in question do not work with either methanol fixation alone or with an acetone:methanol mix (50/50).  Has anyone had issues like this before, in terms of ICC on previously stained slides?  Is there any hope for these markers after the original stain based methanol exposure?  Could I increase my acetone fixation time (currently 10 minutes)?  How about my TritonX concentration (0.1%) or time (10 minutes)?

Thanks in advance for any help that can be provided!  Below, if interested, you'll find an outline of my current working protocol.
----------------
1.  Fix in 100% acetone @ room temp (RT) - 10 min
2.  Dry @ RT - 1 hour
3.  0.1% TritonX100 in PBS - 10 min
4.  PBS Wash
5.  Protein Block - Thermo SuperBlock T20 (PBS) - 30 min (Do not rinse, gently blot off)
6.  Primary Ab - O/N 4C
7.  PBS Wash
8.  3% H2O2 - 15 min @ RT
9.  PBS Wash
10.  Secondary Ab - 30 min @ RT (HRP conjugate)
11.  PBS Wash
12.  DAB Substrate - 10 min (Vector Labs)
13.  H20 Wash
14.  Hematoxylin Counterstain - 2 min
14.  H20 Wash


So have you tried to destain the slides then do the staining. This isn't my field of expertise but I know that antibodies sometimes don't work with Methanol fixation. IF the fix has folded the protein differently then I would do the traditional citrate antigen retrieval with heat and then if that didn't work use a protease AR. My bet is the latter will work. IF its already been fixed with Methanol then I wouldn't further fix it with Acetone. I would do try the AR and see if that helps.

This might help. http://ht.org.ar/immuno33_43.pdf
Also make sure the slides are adhesive before AR.
« Last Edit: October 27, 2011, 04:08:16 PM by Cardio »

Offline MFeirer

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Re: Dealing with pre-stained slides
« Reply #4 on: January 26, 2012, 05:59:25 PM »
Thanks for all of your suggestions.  I never got the markers to work.  My guess is that the MeOH is denaturing my antigen, and that the monoclonals I have are specific to an epitope with tertiary structure.  This would probably explain why my polyclonal still works in this format.  I tried a very large matrix, consisting of various fixatives, AR, and proteases.  AR as it turns out, did work for these markers, but only when previously fixed with a 50/50 mix of Acetone/Methanol.  Not when fixed with only MeOH, as the previously stained slides are.  In any case, we've decided to back up a bit and work with unstained slides.  A bit harder to get, since my pool of old cases get's a lot smaller, but the staining will be easier.  Thanks again for your suggestions!

Re: Dealing with pre-stained slides
« Reply #4 on: January 26, 2012, 05:59:25 PM »