Dear Hadi Taghizadeh, not knowing which staining recipe you will follow - and knowing that almost ALL recipes are written for staining only "sections" (from former paraffin embedded vascular material, I include here the sources of some recipes of Verhoeff-Van Gieson-staining sequence(s) from the www: http://www.ihcworld.com/_protocols/special_stains/vvg.htm,

or, e.g. http://www.med.upenn.edu/mcrc/histology_core/elastin.shtml,

http://www.histosearch.com/histonet/Aug02A/RE.Verhoeff-VanGiesonstai.html

and last but not least: another one:

@http://www.medialabinc.net/chloride-keyword.aspx:

CHEMISTRY: The Verhoeff-Van Gieson (VVG) staining method relies on ferric chloride and iodine to act as a mordant that links hematoxylin dye molecules to tissue components and an oxidizer that converts hematoxylin to hematein. Elastic fibers have the strongest affinity for the hematoxylin-ferric chloride iodine solution which is used to overstain the entire tissue section. Therefore, the elastic fibers retain the dye even after a diluted solution of ferric chloride is used to break the tissue-mordant-dye complex in the other tissue elements. The result is a positive staining reaction that highlights and differentiates elastic fibers from other tissue elements.

Verhoeff-Van Gieson (VVG) Staining - Staining Protocol

Sample type required: Deparaffinized and rehydrated tissue section (3-5 microns) on positively (+) charged slides.

Preferred fixative: 10% neutral buffered formalin (NBF)

Control: Artery or skin

Reagent -Time - Technical Notes

Verhoeff (iron) hematoxylin: 30 minutes. Make fresh; save solution until staining is complete. The ferric chloride in this particular hematoxylin is required as a mordant.

Running water wash: Until solution drains clear

2% ferric chloride solution. Differentiate until black fibers are well defined and background is gray. Review slides microscopically to ensure differentiation is complete. If differentiation is not complete, return to solution for 30 seconds and repeat.

Running water wash: Until solution drains clear

5% hypo (sodium thiosulfate) 1 minute. Removes iodine.

Running water wash 1 minute.

Van Gieson's solution: 5 minutes. Counterstain

Post staining procedure:

Tissue section should be dehydrated with 95% and absolute alcohols followed by two changes of xylene and then coverslip. Expected results: Elastic fibers and nuclei - BlackCollagen - Red. Other tissue elements - Yellow.

I don't know the cause why you have to store the finally stained (i.e. Verhoeff as well as Van Gieson) sections in PBS for 24 hours, but guess that a storage in PBS or just water (tap water) for 24 hrs might retain at least the Verhoeff-staining within your arterial rings (storage @ moderate temperature to 4 degr.C).

At least this would be worth a try (also you could handle this very easily with a kind of unnecessary arterial tissue slice going through the sequence and store it 24 hrs in PBS and see what will have happened afterwards), otherwise you should go back to the roots (cf. Verhoeff-Van Gieson-Staining in:

Pearse, A. G. Emerson (1960). Histochemistry, Theoretical and Applied, 2nd ed. J. and A. Churchill, Londo or perhaps read:

A comparative study of different methods of processing aortic homografts (W. Welch - 1969 ) @ thorax.bmj.com/content/24/6/746.full.pdf

Perhaps, if you don't mind, you could tell us about necessity why to have the whole mount arterial specimens stained in toto and then stored in PBS over 24 hrs??

Best regards and wishes, Wolfgang

Dear Wolfgang,

Kind thanks for your useful reply, I'm trying to get to the sources you provided here.

AS for your information, I'm going to indent stained samples under an AFM to have its mechanical properties profile, to have exact results I need to know where is being exactly stained under AFM. As a result, I need to have my 1-2mm samples stained without standard slicing methods.

The AFM testing instrument is not available instantly after staining and I should keep the stained samples in PBS for around 24 hours after staining.