thank you for all of you. Great respect for all. I already had got some papers about the fat staining in paraffin sections but some points were not clear for me. Simply this method depends on involvement of linoleic acid and chromium trioxide which render the fat resistant to solvents like xylene. The author of this work told me this and sent me a copy of his paper. attached is the paper of this innovative technique.

Hi,

We normally prefer frozen tissue sections over FFPE sections as the dewaxing step on overheating might result in high background staining and xylene treatment dissolves most of the fat depots. I have embedded a link for your assistance. Have a look. Make sure with the Propylene glycol step as it is crucial.

http://www.ihcworld.com/_protocols/special_stains/oil_red_o.htm

If you still have tissue in formalin you may try to freeze and cut a few sections to stain with Oil red. Anything that involves xylene dissolves the fat, so I am afraid it is too late if the only tissue you have is in paraffin blocks because they passed the xylene stage by now.

I agree with Panagiota. Here is a weblink to a related discussion: http://www.ihcworld.com/smf/index.php?topic=3335.0

Xylene dewaxes wehereas the remnants of fat cells remain. So if there's more tissue left and not processed via xylene into paraffin, go to the frozen sections.

I have done the lipid staining on paraffin a number of times before using 0.1% Osmium Tetroxide and the result is similar to the oil red O. Fix tissue normally with 10% formalin for 24 hours and then fix it in 0.1% Osmium Tetroxide for another 24 - 48 hour depend on the tissue thickness. At this point the lipid is fixed, so just proceed with the normal processing, embedding and sectioning. No need to do any further staining, the lipid will be black and the tissue back ground is grey. To convince you can the section with oil red O and you will see the reddish black perimeter around the black lipid droplet. Hope it works for you

We use to fix the tissue in 10% NBF and then cut it in the cryostat as further processing would dissolve the fat.

As others have said, if you have wet formalin fixed tissue that has not been processed into paraffin blocks, you can freeze it and cut cryosections for staining with ORO. You may want to mount the frozen sections on plus or coated slides, as frozen sections of formalin-fixed tissue will not adhere to the slides as well as frozen sections of fresh tissue.

Detection of fats with special staining require frozen sections ONLY.

thank you for all of you. Great respect for all. I already had got some papers about the fat staining in paraffin sections but some points were not clear for me. Simply this method depends on involvement of linoleic acid and chromium trioxide which render the fat resistant to solvents like xylene. The author of this work told me this and sent me a copy of his paper. attached is the paper of this innovative technique.

Use the Adipophilin antibody.

To be honest:

perhaps you should have also a look into a ResearchGate Thread (started 2012-06-28) on this Topic at:

https://www.researchgate.net/post/Is_there_any_suitable_staining_method_protocol_for_lipids_in_paraffin_sections

If your tissue has gone through the tissue processor and is embedded in paraffin then the fat has been extracted and you will not be able to stain for it . That is why fat cells resemble large empty circles.Should have prepared frozen sections then you could of used Oil red O. If you prepare for TEM,you can use the thick plastic sections to see lipid in fat cells.

Dear all,

since I have (had) an interest in such matter I would like to congratulate all the posters here for really good and valauable answers and hints. Honestly I would like to comment on Khuong Le's reply above as follows:

OsO4 naturally plainly IS THE lipid fixative for over a century. As an electron microscopist I am used to use this stuff for more than 33 years now in my profession. So I can tell (what others have published often) that the penetration rate and depth of OsO4 is really low. So if you use only OsO4 at a concentration of 0.1 % and have "big" histological samples (guessing this to be at least 1x1x1 cm=centimeter in dimensions) and you have an "incubation" time of 24-48 hours ("depending on the sample thickness") nevertheless you will get osmicated (=fixed) only a few fat cells (mor/less their vacuoles) situated at the periphery of your specimen. In my (ultrastructural) experience this fixation effect works only 1, at the maximum 2 mm in depth. Using longer "fixation times" can result in elution of tissular components (known as the effect of "prolonged osmication" (to be found in histochemical and ultrastructural basic literature). Usually in T(transmission), as well as in S(canning) EM a 1% buffered (hydrous) OsO4 solution is used, in some special cases recipes using up to 3% OsO4 can be found. This said just to complete other posts. At the end, I would like to enclose the other paper from Tracy&WALIA, published in Virchows Arch (2004) 445:22–26 for your convenience. NB: some major perceptions already have been posted in the former ReseachGate-Thread: https://www.researchgate.net/post/Is_there_any_suitable_staining_method_protocol_for_lipids_in_paraffin_sections .

Best wishes and good luck,

Wolfgang

Thank you so much for all of you and for positive discussion and also for attached relevant papers

I have used Os to stain fat in large samples. The trick is to slice them thin. 10 x 10 x 1 mm as an example. Os fixation impedes its own penetration; so as stated more than a mm or 2 is not going to get fixed. I use 1% OsO4 overnight and rinse in buffer prior to paraffin embedding. It is a great teaching tool to demonstrate that those tiny vacuoles often seen in post mortem liver are not microvesicular fat (because they usually represent swollen mitochondria). I also add a control to the process. I put a thin slice of formalin fixed fat into the process. This also lets you gauge the depth of fixation. If you have not used Os before get help from your local EM person or at least remember, it is nasty stuff and must be tightly sealed in storage and used in a fume hood (volatile and nasty). 

very good staining of lipids in tissue fixed in neutral buffer formalin or slide of paraffin section:

Osmium Tetroxide Paraffin Procedure for Fat

I. The purpose of the stain: Demonstration of fat by a method that allows paraffin embedded of the tissue.

II. The principle of the stain: Osmium tetroxide chemically binds with the fat blackening it in the process. This is the only method for demonstration of fat on paraffin sections.

III. Fixatives: 10% NBF.

IV. Sectioning: Stain the wet tissue with the Osmium tetroxide, then process the tissue. The section must be no thicker than 2 mm or the osmium will not penetrate.

V. Controls: Most tissue contains fats, normally no control is needed.

VI. Reagents:

1. 1% Osmium tetroxide solution: osmium tetroxide & distilled water.

2. 0.5% Periodic acid solution: periodic acid & distilled water.

VII. Procedure:

1. Trim formalin fixed tissue to 2 mm thickness, then wash thoroughly for at least 30 minutes.

2. Rinse the tissue well in distilled water.

3. Place the section in a small quantity of Osmium Tetroxide solution and leave for at least 1 to 2 hours.

4. Rinse slides in two changes of distilled water for 15 minutes each.

5. Differentiate slides by placing them in the 0.5% Periodic acid solution for 30 minutes.

6. Wash slides in tap water for 30 minutes.

7. Process routinely, beginning with 70% alcohol, embed as usual.

8. Cut paraffin sections 4-5 microns, pick on slides and dry as normal.

9. Deparaffinize and hydrate as usual.

10. Counterstain with hematoxylin, Masson Trichrome.

11. Dehydrate, clear and mount slides as normal.

IX. Results:

Fat .............................................................. Black

Other tissue elements ................................. According to method used

 Please, look in attached file

 images of staining with osmic acid in tissue fixed in neutral buffer formalin and proceeding as my above-mentioned method of tissueOsmium Tetroxide Paraffin Procedure for Fat until reaching the final stage of embedding and cutting the blocks on slides and only stain with hematoxylin(osmic acid/hematoxylin).

This method have references attached with my answer beside my work will publish next months

images  (not published yet)--gill of grass carp with rodlet cells

Thanks so much for all the valuable publications which are posted here.

@Hanan Hassan: 

since answers in the ResearchGate -Q&A section usually should meet hopefully scientifically pleasant and valid, but also personal statements and experiences from own work it is deplorable to find out and to know that you use already published information without citing that source(s) - thus you "plagiarize":    

Your reply No 17 above one can find (at least with 95-99% textual identity) at https://quizlet.com/13184659/ht-08-connective-and-muscle-tissue-flash-cards/ :

when the page displays, press >CTRL-F< and insert "Osmium tetroxide stain", then you'll find +/- the whole sequence of sentences in your Re#017.

(BTW, the same content, created by: < Miellee >  - more/less word by word - found also at: https://www.studystack.com/flashcard-1050085  )

So, I regret to say that - if you aren't   user "chaco2589"@ HT 08: Connective and Muscle Tissue /Connective and Muscle Tissue - also the statement in your Re # 019  "as my above-mentioned method of tissueOsmium Tetroxide Paraffin Procedure for Fat" appears as being copied and pasted and therefore your statement seems to be misguiding.

I recommend you to cite the source(s) of "your" method by editing at least your Re#017 or  honestly inform us regarding your authorship of the method (which can be found elsewhere too)

https://www.studyblue.com/notes/note/n/general-fixation/deck/4068240, but the most striking equality / identical text I have found at:

https://de.scribd.com/document/17783222/null, p.26: 

Osmium Tetroxide Paraffin Procedure for Fat

I. Purpose of the stain:

Demonstration of fat by a method that allows paraffinembedded of the tissue.

II. Principle of the stain:

Osmium tetroxide chemically binds with the fat blackening itin the process. This is the only method for demonstration of fat on paraffin sections.

III. Fixatives: 10% NBF.

IV. Sectioning: Stain the wet tissue with the Osmium tetroxide, then process the tissue.The section must be no thicker than 2 mm or the osmium will not penetrate.

V. Controls: Most tissue contains fats, normally no control is needed.VI.

Reagents:

1. 1% Osmium tetroxide solution: osmium tetroxide & distilled water.

2. 0.5% Periodic acid solution: periodic acid & distilled water.VII.

Procedure:

1. Trim formalin fixed tissue to 2 mm thickness, then wash thoroughly for at least30 minutes.

2. Rinse the tissue well in distilled water.

3. Place the section in a small quantity of Osmium Tetroxide solution and leave forat least 1 to 2 hours.

4. Rinse slides in two changes of distilled water for 15 minutes each.

5. Differentiate slides by placing them in the 0.5% Periodic acid solution for 30minutes.

6. Wash slides in tap water for 30 minutes.

7. Process routinely, beginning with 70% alcohol, embed as usual.

8. Cut paraffin sections 4-5 microns, pick on slides and dry as normal.

9. Deparaffinize and hydrate as usual.

10. Counterstain with hematoxylin, Masson Trichrome.

11. Dehydrate, clear and mount slides as normal.

IX. Results:

Fat .............................................................. Black

Other tissue elements ................................. According to method used

Thank you so much Dr. Hanan

Best wishes

Dear Doctor/ Wolfgang H. Muss

Thanks a lot for your comments.I  just used this method with modification.I do not mean this my methods without reference.In the future in my answer, I will refer to reference.

Kind regards

Thank you very much, Dr. Mohamed Alkafafy for the article about lipid fixation.

Kind regards

 Searching RG-Archives of Q&A (e.g.: when logged in  to ResearchGate, paste into URL:   https://www.researchgate.net/search.Search.html?type=question&query=fix++lipid++stain+     or  in the upper menu bar right to JOBS [window with a downpointing arrowhead: leftclick the arrowhead, find the SEARCH function / choose QUESTIONS (left click) and insert your search keywords, in the former case: |   fix  lipid  stain | / RETURN or click icon "magnifier" and you'll get - after some second, a lot of Q&A-threads on the matter you searched / you are searching for.

Then you'll find out also, that the "specific matter" has been questioned (and answered...)  already some times.... e.g.: Unspecific binding issues due to lipid pre-treatment (Tracy and Walia, 2002), what should I do to reduce unspecific binding? (https://www.researchgate.net/post/Unspecific_binding_issues_due_to_lipid_pre-treatment_Tracy_and_Walia_2002_what_should_I_do_to_reduce_unspecific_binding?_tpcectx=search)   or :

https://www.researchgate.net/post/Is_there_any_suitable_staining_method_protocol_for_lipids_in_paraffin_sections?_tpcectx=search

My best wishes and good luck, WM

 . 

Thank you for information.