Dear Özlem,

as stains (in Histology/classical Histochemistry) I found the following:

ex LUNA Lee G, 1992

-carbol fuchsin

-Kinyoun's stain (for Tubercle bacilli AND Lipofuscin)

-Luna-Verfuerth (Fluorescent Method for LIPOFUSCIN & Bacteria)

-Oil Red O

-Ziehl-Neelsen

ex BANCROFT&GAMBLE, 6th Ed., 2008 (Churchill-Livingstone/Elsevier):

-UV-method for Lipofuscins ( cryostat sections 5-10µm, air dried, frozen sections of fixed tissue, paraffin sections) - autofluorescence in UV-light.

The LF's react with a variety of HC and tinctorial staining methods, the most common and useful are:

-PAS

-SCHMORL's ferric-ferricyanide reduction test

-LONG Ziehl-Neelsen method

-Sudan Black B

-Gömori's aldehyde fuchsin technique

-Masson-Fontana Silver technique

-Basophilia, using Methylgreen

-Churukian's silver method,

-Lillie's Nile Blue sulfate Method.

For sure there might be out several other (more/less) specific stains.....but which one of the herein  listed - for now - would you like to have explained or displayed more in detail? ?

best regards, Wolfgang

Thank you very much Wolfgang. I saw also Sudan Black B staining in some articles. If you too, this sharing would be useful as well as H&E. I am not sure to decide which staining method would be excellent to see lipofuscin on brain tissue. There are too many techniques but I should say that I will use stereo investigator for detection.

Dear Özlem, naturally one only knows the/a benefit or „quality“ "effectiveness" and "reliability" of a stain either if done by oneself or having seen color images in a textbook or original article. As an EM-ist I know well the Lipofuscin “granules” (in brain as well, for example, in degenerative muscle fibers or other tissues) which usually can become really areas with striking morphological features too, but admit that I haven’t stained Lipofuscin proper in brain sections for Histology/classical Histochemistry. So perhaps another colleague with “practical” experience will chime in and comment on suitable H-HC-methods for your task (instead of my collecting recipes from Textbooks and Internet-sources providing such a collection here in RG). Guessing most of the Staining techniques mentioned above have been established in the last century.

In LUNA’s book I had the opportunity to compare images of the CARBOL-FUCHSIN and the OIL RED O-method.  

Luna’s Carbol-fuchsin method consists of Kinyoun’s carbol fuchsin, decolorization with 1% acid alcohol and “counterstain” with picric acid 1,18%.

Result: Background:  yellow (cell details hardly visible), Lipofuscin granules/pigment: red.

The Oil-Red-O-method (0.5% in 100% Propylene glycol; to use for a first stain; 85% Propylene glycol to use for a kind of “differentiation/washing”) and “Counterstain” in Mayers Hematoxylin shows in an image included within Luna’s book quite good tissular differentiation between cells as well as Lipofuscin material (and – at least to me – seems really easy to be performed: only some 30 mins – naturally wiothout the staining step in Oil-Red O/Propylene glycol which is 24 hrs and a somehow circumstantial procedure of making the staining solution):

Results: LIPOFUSCIN Material will appear RED, Nuclear Chromatin:  BLUE

If time allows I shall send you as soon as possible a document with copied images as well as the protocol for the staining methods.

Regards,

Wolfgang

Dear Özlem, so far I know Lipofuscin are  small autofluorescending  granuals which  you can detect under a fluorescence- microskop.  To enhance this effect you can depress the autofluorescence of the tissue with sudan black stain.

Procedure: solve 0.3 g sudan black in 100 ml 70 % ETOH; stir it  for  2 h light protected;

stain the vibratome- or cryo-sections  in this solutin for 10 minutes; rinse the sections several times in PBS and mout them in a water soluble mounting  medium.

I found this protocol in our histologiy book:  M. Mulisch, U. Welsch:

Romeis "Mikroskopische Techniken" (2010)

Hope, it will help you. Good luck and best regards

Ute

Thank you Wolfgang. I received your e-mail and the protocols in the articles. They are super helpful, thanks for your support. 

And, thank you Dr. Ute Neubacher. I was wondering if I can apply any staining that I can acquire stereo microscopic results. I saw the protocols (I received  from you and Wolfgang)  require fluorescent techniquies. If you have further directions about steorologic investigation of lipofuscin, please share.

Thank you in advance.

Bests

Dear Özlem, thank you for kind reply.

Naturally no obligation to do it only by FLUOR Microscopy. I only thought It would be perhaps just a rapid option, as far as I was able to see from the articles.

Could you, please, define the term ["I will use stereo investigator for detection" / "steorologic investigation "] a bit more in detail?  Did you mean "stereoscopic" (by means of a viewing your sections) or "stereologic"  (knowing that a requires theoretical knowledge in terms of mathematical calculations (e.g. WEIBEL ff), also see Gorm DANSCHER and Herbert HAUG, + e.g., google ). If the latter, would you mind to send a short line on how you would like to start with that / suchapproach?

Regards and best wishes,

Wolfgang

Dear Wolfgang, I mean, the software that I use is stereo investigator for counting the accumulation, but actually I work on light microscopy basically. I wonder if I stain the brain tissues with H&E staining for monitoring lipofuscin can I detect them on the light microscopy? Does this technique works?

Dear Özlem,

niow I understand that Stereo Investigator is a software package used in LM (cf. http://www.mbfbioscience.com/stereo-investigator). So it would do the job perhaps more sophisticated / more properly than using e.g. or any other imaging software available (f.i. Irfanview or gimp).

So you might find useful also to read the following article:

by Kordower and Collier in: Journal of Comparative Neurology, Volume 502, Issue 5, pages 683–700, 10 June 2007, @ http://onlinelibrary.wiley.com/doi/10.1002/cne.21333/full  (if you can't access the full text, let me know) which also used . I am sure there are out a lot of other papers (e.g. google for:

and for sure the software you mentioned can be used in LM as well as Confocal Microscopy (so I guess it could perhaps be use also for/with  FLUOR MICR).

NB: Regarding your question whether Lipofuscin CAN be detected (and discriminated yourself or by the software) from H&E-stained sections I must admit that I never have done such (staining or measuring/monitoring lipofuscin deposits/areas by means of the mentioned software). So I really don't know if it were possible for an experienced pathologist/cell biologist to discriminate between lipofuscing granules/deposits/patches/areas and other cellular structures stained only by H&E, but to be honest, pointing you perhaps to another possibility using a specific lipofuscin marker:

by Georgakopoulou EA, Tsimaratou K, Evangelou K, Fernandez Marcos-PJ, Zoumpourlis V, Trougakos IP, Kletsas D, Bartek J,  Serrano M, and Gorgoulis VG, in: AGING, Vol 5, No 1 , pp 37-50, 2013 (including also a Recipe for .

 http://www.impactaging.com/papers/v5/n1/full/100527.html or its pdf @ http://www.impactaging.com/papers/v5/n1/pdf/100527.pdf (If you can't download, please, let me know). For sure, if YOU are able (or your imaging system is able) to discriminate Lipofuscin any way from other H&E-stained components in your brain tissue why you shouldn't try this one (at least as a second possibility)? But normally -if "(more) specific stains" are available one would start with a classical histochemical approach, then perhaps with a IHC-method to catch all substrate which can be determined then as being "Lipofuscin".

Regards, Wolfgang