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troubleshooting sectioning of a fresh frozen spleen

holes ice crystals mouse spleen

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4 replies to this topic

#1 rlis

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Posted 11 September 2011 - 11:40 PM

Hi,

I am new in this forum and I hope that I will have answers to my request.
My problem concerns the sectioning of a spleen from a mouse. This mouse is naive, does not bear any tumors and whatever the mouse specie Attached File  spleen.bmp   441.05KB   184 downloadsand the protocol to freeze the slpeen are (snap freezing with or without isopentane, with or without liquid nitrogen, slow freezing with dry ice) I always observe freezing artefacts. The spleen is embedded in OCT and immediately frozen. I joined a photo showing the "white clumps" forming in the organ during sectioning, these white clumps turn into holes after sectioning...
I've never dealt with this troubleshooting in the past and I would appreciate to receive help from you as I need to cut urgently some spleen sections for my project.

Thank you for your help
Rams

#2 Rsm

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Posted 12 September 2011 - 02:23 AM

Have you tired to dehydrate the spleen before freezing? Just put into 20% Sucrose solution at 4C overnight. However, I don't know if that prevents those white areas...
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#3 rlis

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Posted 12 September 2011 - 03:33 AM

Hi,
I've not tried to do that yet...I'll try this option!
Thanks for your help
Rami

#4 PAO_ahac

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Posted 13 September 2011 - 02:29 AM

If you still have problems give a call into a local medium size hospital and see if you can speak with a histologist who should be able to gide you.

#5 rlis

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Posted 13 September 2011 - 04:35 AM

Hi,
Thanks for your advice, this is a great idea.
I'll try to put some sucrose into the spleen for 24hours before snap freezing the sample.
Best regards
Rami




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