May try Toluidine blue

Elyn, interesting question

but unfortunately lacking specific information with regard to concise and sound reply/replies on the failuing of your histochemical staining method.

As of my knowledge and multiple e-files in my e-library  there exist a (huge) variety of Safranin O-recipes for staining not only cartilage and mast cells.

It might be (just only as one thinkable reason) that your BM stem cells don't express proteinaceous material which could stain firmly with the Safranin O you apply/applied (since you wrote that your Alcian Blue ). There also comes into mind the question at which pH you performed the stainings (both of them!).

And naturally (logically) you'll get better answer(s) if you the staining sequence (also: [fixed/unfixed] cryo, paraffin-embedded, in culture, etc. etc.) and recipe you used either for Safranin O as well as the Alcian Blue (including the dye sources/selling companies). Best wishes and regards, Wolfgang

you can use Ethidium bromide:acridine orange (1:1) ratio mix staining

Thanks everyone for the suggestions. Really appreciate it.

Dear Wolfgang,

I think you're  right on the fact that probably  the stem cells did not express the proteinaceous material to strongly stain the safranin O.

I used 0.1% Safranin O and Alcian blue pH 1.0 for cartilage (as stated from the IHCWorld protocol taken here http://www.ihcworld.com/_protocols/special_stains/safranin_o.htm and http://www.ihcworld.com/_protocols/special_stains/alcian_blue_ellis.htm).

My samples were fixed with NBF, praffin embedded.

Ok, dear Elyn,

thank you for the important information. I will need a certain time (and will come back then with suggestions) to dig a bit in my  electronic  files. But would let you know for the moment that it is (well) known for experts (in matrix & matrix proteins - Glycoproteins=GP's - Proteoglyans (PG'S) - Glycosaminoglycans(GAG's)  that (still correctly buffered) formalin/formaldehyde fixation does not retain all possible and real amounts of these substances you might be looking for by final staining of paraffin embdded sections (e. g. I remember some 40%-80% REMOVAL of GAG's & / or PG-constituents by the fixative!) . So if there is only little of such substances to be stained afterwards, you could certainly fail to stain (optically visible) the material you are looking for. I also [in , when I started my "career" as EM-ist with human hyaline cartilage (unborn, perinatal, young, old)] remember that - in cartilage sections - the coloration / color and intensity of staining always was different (with regard to age // ontogenesis, the younger the more “pale”). So you might also know that INCLUSION of Safranine O (e.g. 0.1 to 1% w/v) into the fixative (and all following processing steps -at least until the higher alcohols in dehydration-) in the end will retain more of the molecules you want to localize. IHC-world.com recipes are certainly a valuable source for staining methodology (but I have found out that sometimes not all of the recipes are without any error!) but I do hope that you also stained some control tissue/sections to be sure your method worked correctly.  For now, best wishes and regards, Wolfgang

Dear Wolfgang,

Thank you very much. Would it be possible for you to tell me which paper mentioned about the removal of PG by fixatives? And yes, i did stain the controls and they somewhat worked. So I guess the method is not to be blamed :)

Appreciate much for your kind help and looking forward for further information from your e-files.

Thanks again!

 Dear Elyn, will reply tomorrow or  - at the latest, the day after tomorrow...

Thanks for your patience,

Wolfgang

Dear Elyn,

I am sorry and must apologize for having lost that/this your request / thread which is really interesting....April 13 and the following days were certainly full of other work, I guess and so it happened.. Today I found again this thread and would like to answer as follows:

Please, google for search phrase:   (without < and >! ) There are aprox. 89.600 results, the first 10 or so might answer your most arduing questions...

Especially Király K. et al in   , Histochem J. 1996 Feb;28(2):99-107  ( http://www.ncbi.nlm.nih.gov/pubmed/8737291 ) report about loss of GAG's during FA-fixation (at least 10.6% of GAG's). Also (from their Summary):  "When 0.5% Safranin O was included in the fixative and 4% formaldehyde in the decalcification solution, Safranin O staining of the histological sections increased on average by 13.5%"

It might be that I was wrong in my saying (see my reply #05 above)    "some 40%-80% REMOVAL of GAG's & / or PG-constituents by the fixative!"  because my memory over the last 25-30 years (it was around 1985-1995 when I attended a Congress on Osteology&Matrix Research) decreased./-es...  It might be that 40-80% only holds for very specific substances/glycoconjugates in bone and cartilage (remembering those measures were achieved expressing biochemically specific amino acid values as their contents in evaluating those in the used fixative).

Hoping that meanwhile you've managed and overcome the problem, regards,

Wolfgang

NB: added additionally: cf.: Histochemistry: An Explanatory Outline of Histochemistry and Biophysical Staining

von Richard W. Horobin, Gustav Fischer & Butterworths, 1982 (ISBN 3-437-10700-3 (Fischer):

(p.42): Retention of non-polymeric materials/substances   and Table 8, p.43:

https://books.google.at/books?id=i2meBQAAQBAJ&pg=PA43&lpg=PA43&dq=Loss+of+gag%27s+by+formaldehyde+fixation&source=bl&ots=VYDLYAX5z6&sig=Ksivby8isYawx44TtwczZzDs-aU&hl=de&sa=X&ved=0ahUKEwjfv6WO_bXJAhWKsxQKHQRGCLkQ6AEIKTAB#v=onepage&q=Loss%20of%20gag%27s%20by%20formaldehyde%20fixation&f=false.

Google search for:   

Loss of GAG's by formaldehyde fixation  : approx.200.000 results; 

Loss of GAG's OR Glycoconjugates by formaldehyde fixation  :  ca.29.200 results

Dear Wolfgang,

Thank you very much for your kind assistance, and  I am very  much grateful for your feedback! In fact, I too almost forgot about this question, until I got a notification from you. and for that I thank you again.

Dear Rozlin,

Have you solved the problem at last? If yes, how to solve the problem. Now I meet the same problem with you. Thank you.

Hi Bill,

If I remember correctly, I added 0.6% Safranin O in the fixative and 4% formaldehyde in the decalcification solution (as mentioned in a study by Király K. et al above). I would not say the histo staining looked striking but at least it was better than before. Hope this helps.