Dear Mohd Yusof Mohamad, 

In My experience, 1% Toluidine blue (distilled water) had acceptable results. The old solution have better staining than fresh solution. 1-5 min immersion ( thickness dependence ) in Toluidine blue and first washing with isopropyl alcohol or methanol are recommended.

Good luck ,

Thanks Mr Mehran, do you have an idea why some papers use acetate buffer or ethanol to prepare the reagent?

Dear Mr Mohd Yusof Mohamad, 

your question is not easy to answer because there are some points to be under consideration:

1) cartilage specimen: native, fixed (if yes: which fixative), frozen; which cartilage type.

2) sections: paraffin embedded, frozen?

3) task of your study: only to see cartilage stained blue, stained metachromatically (i.e. NOT blue like using Alcian blue, but presenting in red-violet tones or pink), only morphological overall image,

4) the composition and / or the different but right solute (neutral, acidic, alkaline ) might depend on the expected cartilage's  and its components' staining pattern[*) see bottom line].

5) why it must be Toluidine Blue?....because it might turn out that TB is NOT necessarily the best dye to stain cartilage and its components... etc. etc.  

So I honestly would suggest you to tell a bit more detailed about your personal requirements regarding the study.  Thanking you in advance, BR W.M. 

[*)The staining properties of Toluidine blue - usually a metachromatic dye/stain - are

seriously altered depending on the following parameters:

Adjustments or alterations in any of these parameters will yield very different results.

Only as an example:

A basic pH of 9 will yield:

an intense stain in the extracellular matrix...,   bright blue / purple colour in the growth plate and articular cartilage.

Contrary to that:.

An acidic pH of 4 will stain the nuclei a dark blue / purple colour.]

To achieve such different pH-milieus it is necessary to use either acetic acid (buffer) or basic components/chemicals (like borax/borate or something similar or basic buffer solutions)

Note in addition regarding my reply # 03 above:

It might be advisable to read e.g.

RICHARD J. WASSERSUG (1976): A PROCEDURE FOR DIFFERENTIAL STAINING OF CARTILAGE AND BONE IN WHOLE FORMALIN-FIXED VERTEBRATES. In: Stain Technol. Vol. 51, No. 2 , pp. 131-134

ABSTRACT 

This paper describes a modification of the Simons and Van Horn (1971) procedure for rendering cartilage blue, bone red, and soft tissue translucent or transparent in whole vertebrate specimens.

Alcian blue and alizarin red S are used to stain cartilage and bone respectively.

In our procedure formalin is used as a fixative. This is a significant modification because formalin is the common fixative for museum specimens. This clearing and staining procedure is thus readily applicable to comparative studies in anatomy, embryology and systematic zoology.  

CAN be found @

https://www.researchgate.net/profile/Richard_Wassersug/publication/23075572_A_procedure_for_differential_staining_of_cartilage_and_bone_in_whole_formalin-fixed_vertebrates/links/00b7d52d9a0d9c49a8000000.pdf

NB: The cited reference is only ONE of ancillary but specialized primary literature dealing with the matter. You perhaps will find out a better way or method of staining your cartilage specimens or sections....But - for sure - there are more recent articles out in the wild but they have to be searched for or sought: by digital or practical digging in:  a / in library/-ies, in PubMed, in ResearchGate and in the www.net (Google, or any other search engine you might prefer). And you might find out also why there are so many different recipes.....

B.R., W.M.

Article A Procedure for Differential Staining of Cartilage and Bone ...

Thank you very much Dr Wolfgang for your assistannce

You are welcome, dear Mohd Yusof Mohamad. Best regards and wishes, W.M.