[Histonet] Question about Goldner's Trichrome collagen staining

[Sean Zuckerman]

Dear Histonetters,

I am attempting to use Goldner’s Trichrome to demonstrate bony tissue
formation in human mesenchymal stem cell pellet culture via endochondral
ossification. Based on morphology I see something resembling bony tissue on
the outer surface of the pellets. My sections take Solution A up rapidly (~1
min) but do not seem to de-stain anywhere near as rapidly (15-20 min). And
I’ve found that it takes ~15-20 min in Solution D to obtain reasonable
staining of any collagen. I have found one reference to extended staining
times for Solution C (Orange G/PMA) & Solution D (Light Green) on Histonet,
but the reference was in regards to 15 um sections of ground MMA sections
and I am dealing with 5 um paraffin-embedded sections.  I have also
substituted Fast Green FCF for Light Green in Solution D.  Am I right in
thinking these times for Soln’s C & D are too long? Should I use the
variation with azophloxine? Any advice is greatly appreciated. Thank you for
your time & assistance.



Sincerely,

Sean Zuckerman, PhD

Postdoctoral Research Fellow

Department of Biomedical Engineering

Case Western Reserve University



Extended incubation in Soln’s C & D:

http://lists.utsouthwestern.edu/mailman/htdig/histonet/2008-February/035721.html



I am following the protocol from Gray’s Microtomist’s Formulary (1954):

Weigert’s hematoxylin - 15 min

Rinse in tap water followed by Solution B

Solution A – 5 min

Rinse briefly in Solution B

Solution C – till collagen de-stains

Rinse briefly in Solution B

Solution D – 5 min

Solution B – 5 min

Dehydrate & mount



 Solution A:  100 ml water

                        0.07 g Ponceau Xylidine

                        0.03 g Acid fuchsin

                        0.2 ml Acetic acid, glacial



Solution B:   99 ml water

                        1 ml Acetic acid, glacial



Solution C:  100 ml water

                        4 g phosphomolybdic acid

                        2 g Orange G





Solution D:   100 ml water

0.2 g Fast Green FCF (subbed for Light Green)

                         0.2 ml Acetic acid, glacial