Loss of tissue during staining

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Loss of tissue during staining

Postby Steph_25 » Apr 13 2012 4:35 pm

Hey,
I'm about to do some LCM from mouse eyes. So far I got the cutting to turn out the way I need it but as soon as I try to dehydrate and stain my tissue it will wash off the slide. Those cuts that don't wash off start to crinkle and loosen on the edges. Now I was wondering if anybody had any good ideas or suggestions...
Here's what I'm doing/using:

- Tissues (fixed in PFA) embedded in OCT are cut using a cryostat
- cut sections are transferred to a slide at room temperature and the slide is then placed on dry ice
- the slides are fixed in ethanol (75%) for 30 sec, washed in H2O, stained with Histogene staining solution (20sec.), washed again and then dehydrated (EtOH 75%, 95%, 100%) followed by 5 minutes in xylene and 5 minutes to dry off
- Lcm is performed (Arcturus)

Have you any suggestions for me concerning other slides to use? Or has anybody ever used a desiccator to dry the slides after staining?

Thanks for your help!!
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Re: Loss of tissue during staining

Postby r.rosati » Apr 13 2012 7:11 pm

Hello Steph,
(I'm curious: what system are you using, and which type of slides?)
This is curious - just ten minutes ago I was watching a video I downloaded long ago (for teaching purposes), that's exactly about LCM from mouse eyes.
The video is from here:
http://www.jove.com/video/309/laser-cap ... ian-tissue
I think you should contact the author... he seems a very good candidate to help you!
Best wishes!
Roberto
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Re: Loss of tissue during staining

Postby Steph_25 » Apr 16 2012 1:14 pm

Hey Roberto,
thanks for your reply and the video. The system I'm using is the PixCell IIe LCM system (arcturus) and the slides are superfrost plus slides (VWR). But I'm gonna try some different slides today, hopefully they will work.

Best,
Steffi
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Re: Loss of tissue during staining

Postby r.rosati » Apr 16 2012 7:45 pm

Hello Steffi,
that's a lovely system, I wish I had access to one.
Would you like to take a look at this page and see if anything helps?
http://www.ihcworld.com/_technical_tips ... f_tips.htm
Best wishes,
Roberto
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Re: Loss of tissue during staining

Postby CrowSan » Apr 19 2012 9:51 am

Hi - I had a similar problem a while ago with hydrogels. The simples way is to use coated slides (such as PLL coated or gelatine/chrome/Alum coated slides). In the end I think I used "Superforst Ultra Plus" slides from VWR (no affiliation).
Whilst I still lost the occasional sample the majority stuck down well (Adhesion slides, SuperFrost Ultra Plus® pkt.72
cat. 631-0099 £30.90).
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