Author Topic: Urgent help for cryosection of spleen...  (Read 5362 times)

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Offline Rams

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Urgent help for cryosection of spleen...
« on: September 09, 2011, 09:24:37 AM »
Hi all,

I've been trying to cut some cryosections of mouse spleen but I face with a recurring troubleshooting.
Whatever the protocol used to freeze the spleen in O.C.T (slow freezing using dry ice, snap freezing with liquid nitrogen in the presence or absence of isopentane etc..) I always see white clumps forming into the organ once cut with the cryostat. I tried to play with the temperature of the cryostat chamber  as well as the temperature of the knife but no way to avoid these strange clumps appearing.
I've never seen that in the past while I've cut many times this organ and I don't know whether these "holes" are due to the formation of ice crystals, but in the presence of isopentane I should not see them anymore?
I joined below a photo of the organ to illustrate the problem.


If anyone had any suggestion, protocol or idea to help me solving the problem, I would appreciate a lot!!

Thanky ou very much

Rams

Urgent help for cryosection of spleen...
« on: September 09, 2011, 09:24:37 AM »

Offline gula

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Re: Urgent help for cryosection of spleen...
« Reply #1 on: September 09, 2011, 11:11:46 AM »
I don't see holes, but white matter.
How do the sections appear?

Has your mouse a tumor? Cancer material, nuclei-rich material turns white in cryo. But that should be seen in the section.

just first thought in mind
gula

Offline Cardio

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Re: Urgent help for cryosection of spleen...
« Reply #2 on: September 09, 2011, 11:54:57 AM »
Hi all,

I've been trying to cut some cryosections of mouse spleen but I face with a recurring troubleshooting.
Whatever the protocol used to freeze the spleen in O.C.T (slow freezing using dry ice, snap freezing with liquid nitrogen in the presence or absence of isopentane etc..) I always see white clumps forming into the organ once cut with the cryostat. I tried to play with the temperature of the cryostat chamber  as well as the temperature of the knife but no way to avoid these strange clumps appearing.
I've never seen that in the past while I've cut many times this organ and I don't know whether these "holes" are due to the formation of ice crystals, but in the presence of isopentane I should not see them anymore?
I joined below a photo of the organ to illustrate the problem.


If anyone had any suggestion, protocol or idea to help me solving the problem, I would appreciate a lot!!

Thanky ou very much

Rams
White clumps are different from holes so let us know what exactly you see under a scope. If its holes then it could be to much OCT which will slow down the freezing time. Unless they want a double blind study then you should be able to find out if your dealing with a cancerous spleen.

Offline Rams

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Re: Urgent help for cryosection of spleen...
« Reply #3 on: September 09, 2011, 04:32:38 PM »
Hi,

First I would like to thank you for your help and your interesting suggestions.
Indeed these white clumps appearing in the spleen turn to be holes after sectioning as a quick H&E staining and a observation under the scope do not reveal any cellularity aspect in these white regions.
Moreover, I tried many times to take the spleen fron mice of various species and naive, but the problem persisted. These mice do not develop any tumor and do not receive any treatment.
Your suggestion Cardio can be of interest. I'll try to put less OCT

Offline Rams

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Re: Urgent help for cryosection of spleen...
« Reply #4 on: September 09, 2011, 04:41:17 PM »
Hi,

First I would like to thank you all for your help and your interesting suggestions.
Indeed these white clumps appearing in the spleen turn to be holes after sectioning as a quick H&E staining and a observation under the scope do not reveal any cellularity aspect in these white regions.
Moreover, I tried many times to take the spleen from freshly arrived mice, from various species, naive, but the problem persisted. Importantly, these mice do not develop any tumor and do not receive any treatment.
Your suggestion Cardio can be of interest. I'll try to put less OCT in the tissue, hoping that the clamps will not appear during the sectioning...
Sorry to repeat myself but I tried several methods of freezing without any success for the moment...
The problem seems to concern specifically the spleen as other organs like the lung do not show these curious artefacts.

I am looking forward to receiving other pertinent ideas from you
Thank you for your help
Rams

Offline Cardio

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Re: Urgent help for cryosection of spleen...
« Reply #5 on: September 12, 2011, 02:32:52 PM »
Hi,

First I would like to thank you all for your help and your interesting suggestions.
Indeed these white clumps appearing in the spleen turn to be holes after sectioning as a quick H&E staining and a observation under the scope do not reveal any cellularity aspect in these white regions.
Moreover, I tried many times to take the spleen from freshly arrived mice, from various species, naive, but the problem persisted. Importantly, these mice do not develop any tumor and do not receive any treatment.
Your suggestion Cardio can be of interest. I'll try to put less OCT in the tissue, hoping that the clamps will not appear during the sectioning...
Sorry to repeat myself but I tried several methods of freezing without any success for the moment...
The problem seems to concern specifically the spleen as other organs like the lung do not show these curious artefacts.

I am looking forward to receiving other pertinent ideas from you
Thank you for your help
Rams
Do you cryoprotect with 30% sucrose to remove the water? Liquid nitrogen cooled isopentane is the fastest way to freeze. Search the forum and you can find a protocol.

Offline Rams

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Re: Urgent help for cryosection of spleen...
« Reply #6 on: September 13, 2011, 08:51:19 AM »
Hi,

I do not know whether the antibody I am going to use to stain the sections of the spleen (once I will solve the problems of the white clumps) will work on fixed tissues (PFA, formalin) I wonder if you have ever tried to put your spleen in 30% sucrose O/N at 4C without any previous fixation?
Thank you
Rami

Offline Cardio

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Re: Urgent help for cryosection of spleen...
« Reply #7 on: September 14, 2011, 12:08:37 PM »
Hi,

I do not know whether the antibody I am going to use to stain the sections of the spleen (once I will solve the problems of the white clumps) will work on fixed tissues (PFA, formalin) I wonder if you have ever tried to put your spleen in 30% sucrose O/N at 4C without any previous fixation?
Thank you
Rami
Well you have to test it. I have done what I have suggested but you have to test it with your conditions/antibodies. You don't have to do 30% sucrose overnight you can just do 6 hours or until the tissue sinks. I would try it out and see if thats your problem (ice crystals).

good luck

Offline Rams

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Re: Urgent help for cryosection of spleen...
« Reply #8 on: September 15, 2011, 03:29:26 AM »
Hi,

I tried to cut some sections of the spleen sink into 30% of sucrose and the white clumps still appear during the sectioning. However, these clumps are much smaller once I cut the sample very slowly with the handwheel...I wonder if the real problem is the speed of cutting rather than the formation of ice crystals...I tried again to cut another organ and it was fine. The spleen seems to be capricious

Cheers,
Rams

Offline Dunia

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Re: Urgent help for cryosection of spleen...
« Reply #9 on: October 28, 2011, 05:45:30 AM »
Hi,
I have cut mouse spleen many times and it worked fine: snap frozen in liquid nitrogen with or without OCT, then stored at -70C, cut with the cryostat temperature -18-20C.
We do usually get this image of red organ with white clumps, but consider it normal since in spleen lymphoid regions containing B- and T-cells are organized into white pulp with the red pulp surrounding them. As I can judge by immunofluorescent staining of the spleen cryosections red pulp is more dense and has higher autofluorescence due to erythrocytes, white pulp is less dense and on the unstained control may give you an impression of holes due to much less autofluorescence in contrast with the red pulp. Unfortunately it is a quite often artifact due to sectioning when on some sections (not on the entire series) white pulp smudges or even fells out (very rare). I suppose that this artifact can be explained by the difference in the density and structure of those two regions of the spleen: the red pulp and the white pulp.
I think you should not be scared of these "white clamps" , just examine the sections under the microscope more precisely and may be stain with something that surely works well, for example an antibody against B or T lymphocytes.
« Last Edit: October 28, 2011, 05:55:48 AM by Dunia »

Re: Urgent help for cryosection of spleen...
« Reply #9 on: October 28, 2011, 05:45:30 AM »