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Dead staining... some samples work others don't PLEASE HELP!

Dead staining... some samples work others don't PLEASE HELP! - Immunofluorescence Forum

Dead staining... some samples work others don't PLEASE HELP! - Immunofluorescence Forum. Talk, discuss and post questions on immunofluorescence techniques including paraffin, frozen sections or cultured cells.


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Old 03-01-2010, 04:03 PM
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Default Dead staining... some samples work others don't PLEASE HELP!



Hello all,

I'm currently staining human bone marrow for:

PDGFR(alpha)
PDGFR(beta)
VCAM-1
STRO-1
DAPI

That's a lot to stain for at one time I know, but so far it's staining well... but only on some samples. Other samples (stained along: same jar, ab mix, at the same time, ect.) do not stain at all. Here's the kicker... even DAPI does not stain on these slides.

Does anyone have any ideas about what could be going on?

I get that samples from a histology lab in a hospital, so unfortunately I have no way of knowing how the slides were treated post biopsy. The samples are fixed in paraffin and cut in the histology lab. They come to me pre melted.

Here's the protocol I'm using:

1. xylenes 2x 5 min.
2. 100% ethanol 2x 3 min.
3. 90% ethanol 2x 3 min.
4. 80% ethanol 2x 3 min.
5. H2O 2x 1 min.
6. Antigen retrieval: 4.5 min. in sodium citrate buffer @ 95C ("happy" medium between optimal time for all antigens)
7. PBS 2x 1 min.
8. Block 2% serum for 1 hour @RT
9. Primary antibody (optimal concentrations in PBS) overnight @4C
10. PBS 2x 5 min.
11. Secondary antibodies (optimal concentrations in PBS) for 1 hour at RT.
12.PBS 2x 1 min.
13. tap H2O 1 min.
14. mount using Vectashield (with DAPI)

Image using confocal.

I spent so much time optimizing antibody concentrations and antigen rescue times. The slides I used to optimize everything were all from the same sample, and it works great. When I moved to patient samples, only 1/3 of the samples stained for anything (normal or disease... doesn't matter).

The key here is that even the DAPI does not work. Meaning it's not an antibody/antigen issue. And, all of the slides have what looks like good nuclei.

Any ideas are appreciated. Iím completely stumped.
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Old 03-01-2010, 05:17 PM
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Default Re: Dead staining... some samples work others don't PLEASE HELP!

Generally speaking, heterogeneity like you describe come either from preparation mistakes or from having samples from different sources that need to be treated differently. It doesn't sound like the latter case is your issue (if it were, you'd see consistency between what works and what doesn't - i.e. controls are always screwed up).

Unfortunately, the only real solution here is to be extra anal about your prep, to ensure every sample gets treated exactly the same. That said, the DAPI staining is odd and brings up a few questions:

1) Are there still cells in the samples that don't stain? Its possible that over fixing or dehydration may destroy your cells, hence no staining.

2) Are you sure the parafin is being completely removed from all samples prior to staining? Parafin can prevent antibody/dye access if it covers the surface you are staining.

That's all I can think of.

Bryan
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Old 03-01-2010, 05:49 PM
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Default Re: Dead staining... some samples work others don't PLEASE HELP!

Bryan's got the right ideas.

I especially would zero in on the de-paraffinization.
Do a 95C melt step for 5 min, followed by 3 x5min xylene dips, that should take care of it.

You can also increase the hydration, I would hydrate in a series from 95-50% EtOH, for 5 min each to be extra safe.

If that still fails for those stubborn samples, then you can eliminate paraffin or hydration as possible factors. I would then take a look at fixatives.
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Old 03-01-2010, 06:21 PM
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Default Re: Dead staining... some samples work others don't PLEASE HELP!

And I know this isn't what you want to hear, but when parafin doesn't work there is always frozen sections...

Bryan
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Old 03-01-2010, 06:55 PM
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Default Re: Dead staining... some samples work others don't PLEASE HELP!

Thanks guys. All of it is good advice. My main problem is I don't have access to the samples prior to the histology lab in the hospital. Ergo, I have no idea if the preparation of the tissue prior to paraffin fixation is consistent. The samples are from a rare type of leukemia and have been collected in a library. Some samples are 1 month old, others 10 years. However, there is no correlation between the age of the samples and the success of the staining.

I agree that improper paraffin removal is the most likely culprit. I’ve tried to increase xylenes exposure but to no avail. As for heating the slides first, the slides are given to me pre-melted. I’ve tried to heat them again, but that doesn’t help either.

I’ll try increasing the rehydration step down to 50% ethanol. That may help a lot.

In answer to Warthaug’s question, the cells are definitely intact. Nuclei are clearly visible on a brightfield.

As far as fixatives, I have no idea how the pathology folks treated the samples prior to the paraffin. I was told that paraffin was the only fixative.
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Old 03-01-2010, 07:53 PM
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Default Re: Dead staining... some samples work others don't PLEASE HELP!

Quote:
Originally Posted by bigdmajor View Post
I’ll try increasing the rehydration step down to 50% ethanol. That may help a lot.


As far as fixatives, I have no idea how the pathology folks treated the samples prior to the paraffin. I was told that paraffin was the only fixative.
I recommend Davidson's fixative, [Only registered users see links. ]

especially over formalin/paraformaldehyde (which is most popular in my experience).
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Old 03-10-2010, 09:30 PM
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Default Re: Dead staining... some samples work others don't PLEASE HELP!

Found the problem.

The path lab uses a particularly harsh de-calcification solution. I found out that a lot of the samples were processed on Friday, and LEFT OVER THE WEEKEND in de-calcification solution. Well, what do you know... these happen to be the samples that don't stain for anything

If only someone in our lab was in charge of processing the samples...

Sigh,

Well thanks for all the suggestions, but unless anyone knows of a way to undo entropy and reverse catastrophic HCl damage, I think I'll be trashing 2/3 of these samples.

cheers everyone
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