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Author Topic: FFPE White Adipose Tissue (WAT)  (Read 13746 times)

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Offline Siv

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FFPE White Adipose Tissue (WAT)
« on: October 16, 2012, 07:42:31 PM »
I'm having issues with adherence of my FFPE WAT on microscope slides for immunofluorescent (IF) staining.  The amount of tissue loss is highly variable.  As early as deparaffinization step, I'm observing small amount of tissue 'wiggling' off the glass.  I fear that I continue to lose tissue every step of my protocol (i.e. antigen retrieval, washes, etc.).

Some protocols recommend incubating the sections at 60 degrees C for few hours.  Are there any downsides for IF staining if I incubate WAT at 60 degrees C?  The loss of tissue varies from sample to sample. 

I am placing 8-10 micron sections on positively charged glass slides (Thermo's Shandon slides (#99-910-01)), and I'm using Leica's Sta-On to help with the adherence.

FFPE White Adipose Tissue (WAT)
« on: October 16, 2012, 07:42:31 PM »

Offline gula

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Re: FFPE White Adipose Tissue (WAT)
« Reply #1 on: October 17, 2012, 11:52:50 AM »
For adipose tissue it's very important that it is properly processed. First good fixation, then long infiltration with 100% ethanol and xylen (or similar) to remove the fat. Then long infiltration with paraffin is of advantage.
With sectioning it's important, that any residual water is removed under the section, when the slides do only airdry. The section dry from the outside and hold water between glass and tissue, that inhibits correct adherence. Therefore drying at melting point of paraffin helps evapuration of the water and adherence of the tissue.
The sensibility of the antigen depends on the antigen not on the detection-technique. Best practice would be performing your analysis with positiv controls to proofe your protocol. For example we perform IF on FFPE kidney for immunoglobulins. These slides are dried at 60°C.
gula

Offline Siv

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Re: FFPE White Adipose Tissue (WAT)
« Reply #2 on: October 17, 2012, 05:55:03 PM »
Gula, thank you.

Offline Siv

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Re: FFPE White Adipose Tissue (WAT)
« Reply #3 on: October 30, 2012, 11:18:05 AM »
Gula,

Is it possible to fix tissue overnight, then freeze them away until they can be paraffin embedded? 

Offline gula

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Re: FFPE White Adipose Tissue (WAT)
« Reply #4 on: October 31, 2012, 04:01:01 AM »
Why do you want to freeze the tissue? Freezing in aeqeuous solutions brings very much cristalformation and therefore tissue-disruption. If you really want to do that, you would have to displace the fixative through a sucrose-freezing-medium, that would take much time (I think, never done). And that has only sense, if you want to produce frozen sections. And frozens are only necessary, if you fear to loose any tissue-compound through paraffin-processing.

Usually there is no negative effect, if you leave the tissue for a longer period in NBF until paraffin processing. If you fear to get more autofluorescence through longer formalinfixation, you can put the tissue in 70-80% ethanol until paraffin-processing, but autofluorescence would never be depressed once formalinfixed.
hope this helps
gula

Offline immuno

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Re: FFPE White Adipose Tissue (WAT)
« Reply #5 on: October 31, 2012, 07:54:01 AM »
Hi:

I had the same problem and I solved it treating the slides  with poly-L-Lisine and after that with vectabound. Itīs a expensive method but he "double adhesive" in the slides really helps.Be sure that you dry the sections properly.

Immuno

Offline Siv

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Re: FFPE White Adipose Tissue (WAT)
« Reply #6 on: October 31, 2012, 11:59:51 AM »
There are publications that state that over fixation has the potential to masking antigens for IF or IHC.  They stated that antigen retrieval can be ineffective in rescuing masking cause by fixation.  Because we process one mouse a day, the lab waits until a large collection of tissue has been accumulated before sending it out to be paraffin embedded.  The tissue can sit in formalin for a long period of time before being processed.  I'm trying to think of an intermediate stage that prevents over fixation.  I'll look into placing the tissue in 70-80% ethanol until paraffin-processing.  Would sitting in 70-80% ethanol for a week or longer have any deleterious effect on downstream assays, such as IF or IHC?

Immuno, thank you for you recommendation.  What appears to work for our WAT is placing FFPE section on positively charged slides, Thermo-Shandon slides (#99-910-01), and have Leica’s Sta-On (3803105) in the water bath.  We place the slides on Richard-Allan Scientific Micro Slide Holder to prevent the water from being trapped between the paraffin sections and glass, and then place our slides in a 37 degree incubator for a couple of hours to thoroughly dry.  I'm having success with the tissue staying on.

Offline gula

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Re: FFPE White Adipose Tissue (WAT)
« Reply #7 on: November 01, 2012, 03:38:29 AM »
I see your point, but have no practical experience in holding tissue in ethanol. In clinical lab the demonstrated antigens should be able to be shown after a week fixation with no altering of the staining protocol, but are still stainable after 4-6 weeks, or years. It depends very on the special antigen, you have to demonstrate.
Is it very hard to find a histolab, that does the paraffinprocessing for you? The paraffin blocks could be stored very long until cutting and staining.
The publications about overfixation sometimes put all antigens in one pot. And now the trend is to emphazise good fixation with no delay, and optimize antigentretrieval and IHC-protocol.
Fatty tissue tends to be fixed slowly, because penetration is hampered by the fat. If I can fix it two days, I would do it. Your dilemma is, that you see the results only afterwards.
Perhaps someone with more experience in this field is willing to help you.
gula

Offline CanuckPhD

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Re: FFPE White Adipose Tissue (WAT)
« Reply #8 on: November 02, 2012, 02:04:22 AM »
If we have a multiple batchs of rodent tissue (brain, gut, spleen but not fat) we fix for the standard time (24 hours usually), then place in PBS. The tissues are fine and there is no increase in fixation. Then we wash well in water before processing. We have never had any issues with antigen retrieval and the tissue quality is good.

Offline Siv

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Re: FFPE White Adipose Tissue (WAT)
« Reply #9 on: November 27, 2012, 04:57:12 PM »
Thanks CanuckPhD, we'll consider that protocol.  How long do you guys normally keep your tissue in PBS post fixation?

Offline CanuckPhD

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Re: FFPE White Adipose Tissue (WAT)
« Reply #10 on: November 28, 2012, 03:33:22 AM »
We have had no problems with keeping the tissue in PBS for up to 1 week after fixation.

Re: FFPE White Adipose Tissue (WAT)
« Reply #10 on: November 28, 2012, 03:33:22 AM »