| From: | "Starkus, Laurie" |
This message is in MIME format. Since your mail reader does not understand this format, some or all of this message may not be legible. ------=_NextPartTM-000-0d4658b5-9548-4bd9-89f2-649390a09b21 Content-Type: multipart/alternative; boundary="----_=_NextPart_001_01C3BFE4.CB4066B0" ------_=_NextPart_001_01C3BFE4.CB4066B0 Content-Type: text/plain; charset="ISO-8859-1" MinTan- It sounds like you have a fold of frozen section mountant over the center of the section. The mountant is water soluble and needs to be removed. It is not soluble in ethanol. So, be sure that you run your sections under water and this should solve your problem. -----Original Message----- From: Tan, MinHan [mailto:MinHan.Tan@vai.org] Sent: Thursday, December 11, 2003 12:48 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Frozen sections and loss of nuclei? Hi there Histonetters, I would like to seek your advice about a problem I am having with frozen sections. I am working on some frozen tissue dating back to about 1998, and performing IHC on parathyroid tissue with p27 antibody. I use the DAB-ABC method. I have noted that certain samples (and particular samples only) exhibit loss of nuclei in the central region (an amorphous blob of tissue extending to near the margins, about 70-80% of the specimen) (no staining with hematoxylin either). Any nuclei that stain with my IHC in that area are very faint, and there is no hematoxylin counterstained nuclei in the entire region. Nuclear staining seems to be fine near the margins - of course it can be argued that these are artifacts. Where there are folds of tissue, both nuclear and background staining is prominent. Other samples of the same type of tissue seem to stain OK throughout the tissue. No problems with paraffin-embedded tissue at all, the nucleus stains fine in every sample I have tried. It is unlikely to be due to underfixation, since I immerse my 5 micron unfixed slide in neutral buffered formalin for 30 min prior to IHC. I see it regardless of whether I perform HIER on my frozen sections or not. (yes, I do perform HIER on my frozen sections) I wonder if the following explanations are plausible: (a) tissue has degraded - but it would not make sense for degradation to take place from inside out. (b) sections may be too thin for some reason? (mine are 5 micron) - but this might explain why only the borders and folds stain. Thank you! Min-Han Tan Van Andel Research Institute _____ This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential information. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient(s) please contact the sender by reply email and destroy all copies of the original message. Thank you. _____ ------_=_NextPart_001_01C3BFE4.CB4066B0 Content-Type: text/html; charset="ISO-8859-1"Message MinTan- It sounds like you have a fold of frozen section mountant over the center of the section. The mountant is water soluble and needs to be removed. It is not soluble in ethanol. So, be sure that you run your sections under water and this should solve your problem.------_=_NextPart_001_01C3BFE4.CB4066B0-- ------=_NextPartTM-000-0d4658b5-9548-4bd9-89f2-649390a09b21-- _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet-----Original Message-----
From: Tan, MinHan [mailto:MinHan.Tan@vai.org]
Sent: Thursday, December 11, 2003 12:48 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Frozen sections and loss of nuclei?Hi there Histonetters,I would like to seek your advice about a problem I am having with frozen sections.I am working on some frozen tissue dating back to about 1998, and performing IHC on parathyroid tissue with p27 antibody. I use the DAB-ABC method.I have noted that certain samples (and particular samples only) exhibit loss of nuclei in the central region (an amorphous blob of tissue extending to near the margins, about 70-80% of the specimen) (no staining with hematoxylin either). Any nuclei that stain with my IHC in that area are very faint, and there is no hematoxylin counterstained nuclei in the entire region. Nuclear staining seems to be fine near the margins - of course it can be argued that these are artifacts. Where there are folds of tissue, both nuclear and background staining is prominent. Other samples of the same type of tissue seem to stain OK throughout the tissue. No problems with paraffin-embedded tissue at all, the nucleus stains fine in every sample I have tried.It is unlikely to be due to underfixation, since I immerse my 5 micron unfixed slide in neutral buffered formalin for 30 min prior to IHC. I see it regardless of whether I perform HIER on my frozen sections or not. (yes, I do perform HIER on my frozen sections)I wonder if the following explanations are plausible:
(a) tissue has degraded - but it would not make sense for degradation to take place from inside out.(b) sections may be too thin for some reason? (mine are 5 micron) - but this might explain why only the borders and folds stain.Thank you!
Min-Han TanVan Andel Research Institute
This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential information. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient(s) please contact the sender by reply email and destroy all copies of the original message. Thank you.