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Author Topic: water water everywhere  (Read 4182 times)

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Offline DPrater

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water water everywhere
« on: September 08, 2011, 07:55:56 PM »
Hi all -

I'm pounding my head against the wall on this one. I'm doing routine dehydration of slides following standard IHC like I've done a million times - graded ethanol followed by xylenes and then mounting in cytoseal - and I'm having a very unusual and very frustrating problem:

I can't seem to remove all the water from my slides. Enough water is entering the xylene bath following the 100% EtOH bath that the xylenes become cloudy white every time. If I remove a single slide from the 100% EtOH bath and wave it around in the air I can see water beading up on the slide as the EtOH evaporates. And it's a lot of water. I'm using new ethanol - I've tried a number of different ethanols, all histo grade - and for whatever reason I can't seem to remove the water at all.

I've searched the net, this forum, asked around, and three of us here in the lab have no idea where the water is coming from nor how to get it out of the system. We're really banging our heads against the wall on this one. Does anyone have any advice?

Thank you all for your help!

Daniel
UC Davis
dnprater at ucdavis dot edu

water water everywhere
« on: September 08, 2011, 07:55:56 PM »

Offline Cardio

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Re: water water everywhere
« Reply #1 on: September 09, 2011, 12:06:37 PM »
Hi all -

I'm pounding my head against the wall on this one. I'm doing routine dehydration of slides following standard IHC like I've done a million times - graded ethanol followed by xylenes and then mounting in cytoseal - and I'm having a very unusual and very frustrating problem:

I can't seem to remove all the water from my slides. Enough water is entering the xylene bath following the 100% EtOH bath that the xylenes become cloudy white every time. If I remove a single slide from the 100% EtOH bath and wave it around in the air I can see water beading up on the slide as the EtOH evaporates. And it's a lot of water. I'm using new ethanol - I've tried a number of different ethanols, all histo grade - and for whatever reason I can't seem to remove the water at all.

I've searched the net, this forum, asked around, and three of us here in the lab have no idea where the water is coming from nor how to get it out of the system. We're really banging our heads against the wall on this one. Does anyone have any advice?

Thank you all for your help!

Daniel
UC Davis
dnprater at ucdavis dot edu
Have you tried changing the xylene and have you tried a different companies of ethonal. If your at a university a lot of the time the ethanol can come from one vender and hence a bad batch of ethanol, going to another  labs ethanol may not be the solution. The other thing is to extend the ethanol dehydration steps to a excessive amount. NOrmal tissue should dehydrate relatively fast but thickness and type can affect it.

Offline DPrater

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Re: water water everywhere
« Reply #2 on: September 09, 2011, 01:12:46 PM »
Thank you, yes, we did change xylene vendors, and we changed between two different EtOH companies (from house-blend to Fisher histo-grade EtOH). I'm presuming these were from different manufacturers... otherwise the world has a big water contamination issue and our industrial processes are all going to grind to a screeching halt in the near term (which would be entertaining but doesn't help my IHC problems). Anyway...

Thank you. I'm trying a longer dehydration step in 100% EtOH. Overnight this time. They're fairly dense tissues - thymus, spleen, etc, with lots of nuclei, but fact is that the water really doesn't appear to be localizing to the tissue. It's like the water is in the EtOH and when I evaporate the slide by waving it around in the air the water just beads up wherever - not localized to the tissue. If I had to guess I'd say my 100% EtOH is acting like 70% EtOH.

I think I'll be getting an alcohol hydrometer. What do you guys think?

Offline Cardio

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Re: water water everywhere
« Reply #3 on: September 09, 2011, 01:22:37 PM »
Thank you, yes, we did change xylene vendors, and we changed between two different EtOH companies (from house-blend to Fisher histo-grade EtOH). I'm presuming these were from different manufacturers... otherwise the world has a big water contamination issue and our industrial processes are all going to grind to a screeching halt in the near term (which would be entertaining but doesn't help my IHC problems). Anyway...

Thank you. I'm trying a longer dehydration step in 100% EtOH. Overnight this time. They're fairly dense tissues - thymus, spleen, etc, with lots of nuclei, but fact is that the water really doesn't appear to be localizing to the tissue. It's like the water is in the EtOH and when I evaporate the slide by waving it around in the air the water just beads up wherever - not localized to the tissue. If I had to guess I'd say my 100% EtOH is acting like 70% EtOH.

I think I'll be getting an alcohol hydrometer. What do you guys think?

The only other thing I can think of is if some tech screwed up the dilutions in the gradient. I have seen students do a 30% Ethanol dilution thinking it was a 70%.

Worst case you can air dry after the staining in fume hood for 1-2 hours then mount with cytoseal until you figure it out.
« Last Edit: September 09, 2011, 01:24:21 PM by Cardio »

Offline DPrater

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Re: water water everywhere
« Reply #4 on: September 09, 2011, 01:32:19 PM »
Won't air-drying in the fume hood screw up the morphology? If not, then cool. I can do that. It's the no-work way of getting stuff done, and I'm down with that.

Anyway, yeah, I know about the 30%/70% screw-up because I've done it before, but this time the EtOH is straight from the EtOH jug. It's actually straight from a number of different EtOH jugs because I've been working through different EtOH vendors trying to have this water-contamination issue not happen.

I'll probably make a trip to the Chem dept and see if those guys have a hydrometer I can use for the day. Might as well test my stock. And perhaps keep testing it, to see when/if it changes constitution.

Thanks again for your help. It's been very helpful talking this out. I'll let you know what I find out - please keep updating me with whatever pops into your brain because my brain is now empty on this subject.

Cheers,
Dan

Offline MT Scientist

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Re: water water everywhere
« Reply #5 on: September 09, 2011, 01:45:48 PM »
How long are the slides kept in the previous ethanol steps?  It seems with thick tissues, it may need a longer time to soak. 
Also, 100% EtOH can suck water from the air if the container doesn't seal well.  You may also need to follow a stringent rotation schedule for your reagents.

Offline DPrater

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Re: water water everywhere
« Reply #6 on: September 09, 2011, 02:27:35 PM »
The prior EtOH steps were a couple minutes each. Maybe two minutes each, and we're using two baths each of 70%, 90%, and 100%. And I do a bit of dipping to keep things nice and mixed up.

A colleague reminded me today that there have been a number of ghostly manifestations in our lab. Last year we had a massive bleach corrosion episode in one of our cell culture biosafety cabinets while we were all away on a retreat and the culprit has never come forward... this particular EtOH issue might be supernatural. I do sincerely hope that my IHC issues do not require the employment of a priest.

Thanks for your help - I'm on my way to the enology department.

Offline Cardio

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Re: water water everywhere
« Reply #7 on: September 12, 2011, 02:29:09 PM »
Won't air-drying in the fume hood screw up the morphology? If not, then cool. I can do that. It's the no-work way of getting stuff done, and I'm down with that.

Anyway, yeah, I know about the 30%/70% screw-up because I've done it before, but this time the EtOH is straight from the EtOH jug. It's actually straight from a number of different EtOH jugs because I've been working through different EtOH vendors trying to have this water-contamination issue not happen.

I'll probably make a trip to the Chem dept and see if those guys have a hydrometer I can use for the day. Might as well test my stock. And perhaps keep testing it, to see when/if it changes constitution.

Thanks again for your help. It's been very helpful talking this out. I'll let you know what I find out - please keep updating me with whatever pops into your brain because my brain is now empty on this subject.

Cheers,
Dan

Air drying can give you artifacts but it depends on the tissue, slide, and the stain. My bet is that you won't notice that much of a difference.


Re: water water everywhere
« Reply #7 on: September 12, 2011, 02:29:09 PM »