Paraffin Embedding

pundir

New member
Nov 16, 2015
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#21

Hi guy


i think you should check all the reagents whether they are pure, or else i have a resource by which you can make out whether the alcohol you are using is absolute or not as it seems water is the main culprit behind the prob.


"How can I determine whether used "absolute" alcohol is still OK for the last stage of dehydrating specimens or slides ? " www.biologicalstaincommission.org/index.html



 

peter36

New member
Nov 16, 2015
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#22

improper dehydration leads to improper clearing and improper wax penetration. Tissue will appear soft and tend to explode on the water bath. Crunchy tissue is usually caused by extended xylene times on H@E staining the edges will appear very pink with eosin staining "cooked"
 

sumitnbrc

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Nov 16, 2015
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#23
you can follows

1 -24 h in melted paraffin 58deg cel.

2- 30min in melted paraffin 60 deg cel.

3- 15 min in melted paraffin 60 deg cel .
 

Myofib

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Nov 16, 2015
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#24
My student is having this problem today, soft tissue (human skin) that explodes in the water bath. We are going to melt and return it to 95% o/n then finish the dehydration slowly. It is being done by hand. I have done this a number of previous times without any trouble. I think the xylenes are contaminated.
 
Sep 16, 2016
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#26

Hello,

in our lab we established paraffin embedding in thicker parts of organs yet we have troubles with freshly cut 300um rat brain slices.

Firstly, the slices get wrinkled. Therefore I cover them during the fixation (24h) and uncover them during dehydration until xylene. I can see they are getting wrinkled during the hydration yet I don't want them to cover with anything because the process might not be efficient then. At that point (during xylene clearing and wax infiltration) I keep them in cassettes filled with sponges. But in the end, they are still a bit wrinkled. I can see it when cutting the slices, there are holes in my 10um slices. Since I started to use the sponges it got better but still not enough.

Secondly, the slices get fragile and brittle almost immediately when I put them in xylene. The xylene appears transparent so I don't think the water is the problem.

Thirdly, when I embed the slices, I put the slices in a hot wax and transfer the mould on a cold surface so that the wax hardens very quickly. Has anyone tried with pressing the slices against the mould?

The protocol is fixation overnight, 3x PBS wash, 2x 30min 50% etOH, 70% etOH overnight, 95% etOH/ 5% methanol 1h, 100% etOH 3x30min, 50% etOH/50% xylene 30min, xylene 2x 1h, first wax 1h, second wax overnight, embedding. The fixation lasts for 24h but I shortened the rest of the protocol by half and I don't see any difference. At least in these thin slices.

Hope anyone has some experience in this,

best wishes,K