I need to determine lipid accumulation in liver. I plan to fix the tissue in 10% formalin. Should I embed the fixed tissue in paraffin, or cryopreserve the fixed tissue to prepare sections ?
Hi
I do not think so, because in the treatment with Xylene in order to achive the embedding of the tissue with paraffin you could lose a large proportion of the lipids that you want to detect. In this regard, you will need to work on frozen sections obtained with a cryostat. If you can work with cryostat, you could apply the stainings of Sudan III or Sudan IV and the Sudan Black and Oil Red O for the study of lipids.
Good luck, and if yo need to know the protocol do not hesitate to contact me
Hi
I do not think so, because in the treatment with Xylene in order to achive the embedding of the tissue with paraffin you could lose a large proportion of the lipids that you want to detect. In this regard, you will need to work on frozen sections obtained with a cryostat. If you can work with cryostat, you could apply the stainings of Sudan III or Sudan IV and the Sudan Black and Oil Red O for the study of lipids.
Good luck, and if yo need to know the protocol do not hesitate to contact me
Hi too,
guessing that you will have to analyse micro-/ macrolipidic (experimental or from patients liver biopsies?) alterations in (human?) liver ....
I just would like to point you to similar threads on RG with a lot of information concerning topic "lipid localization / analysis and staining for"..(paraffin embedding vs. / or cryosections) (in case you would like to read about):
http://www.researchgate.net/topic/Histology/post/Is_there_any_suitable_staining_method_protocol_for_lipids_in_paraffin_sections?
https://www.researchgate.net/post/Does_anyone_have_ideas_about_lipid_fixation_for_fat_staining_in_paraffin_sections?
https://www.researchgate.net/post/I_want_to_use_Histochemistry_for_detection_of_protein_and_lipids_Can_anyone_suggest_a_procedure_without_using_Cryocut?
Good luck (and I assume that Marcello can/will provide cryo-methods/staining recipes if you demand them...) and best wishes.
Hi,
Very conceptual question with conceptual answ er
Lipids are extracted by the organic solvents, during paraffin embedding, thus the lipid droplets remain as "holes" in the cells. Some lipoproteids could remain in the sample, due to cross linking of the protein part from the fixative, resulting in possible detection by means of IHC. One can use the extraction phenomenon as a surrogate for lipid quantification, e.g. more or bigger "holes" - more lipids.
In general, lipid staining is performed on unfixed or cross-linker (formaldehyde, glutaraldehyde) fixed samples, where the lipids will not be extracted. One can stain further with oil red, sudan black, nile red etc
Cheers and good luck!
you can work with frozen section, staining with Sudan III or Sudan IV and the Sudan Black and Oil Red O for the study of lipids.
Thanks for all the answers !
@Marcello: Can you provide me the protocol ?
You could post-fix the lipids with a solution of 1% Osmium Tetroxide before processing into paraffin.
Also see the following article, although I have no personal experience with it.:
Histopathology. 2002 Jul;41(1):75-9.
A method to fix lipids for staining fat embolism in paraffin sections.
Tracy RE1, Walia P.
Author information
1Department of Pathology, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA. [email protected]
Abstract
AIMS:
To develop a method to preserve lipids in formalin-fixed tissues for staining in paraffin sections, and to illustrate its use in lung and brain of a fat embolism case, and in examples of fatty liver and atheroma.
METHODS AND RESULTS:
A saturated solution of linoleic acid in 70% ethylene glycol was prepared and tissues were exposed to this for 3 days at 56 degrees C. These tissues were treated with 2% chromic acid at 4 degrees C for 24 h followed by 24 h in 5% sodium bicarbonate, with appropriate rinsing between solutions. Paraffin sections of these tissues were stained with a lipid-soluble dye such as Oil Red O. Examples of fat embolism, fatty liver, and atheroma were shown photographically as illustrations of expected results.
CONCLUSIONS:
The demonstration of fat embolism with good quality tissue detail is made practical by the method, which is convenient and inexpensive. The method appears to be generally applicable to tissue lipids of various sorts, as exemplified by adipose tissue, fatty liver, and atheroma.
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