RE: [Histonet] restaining of hematoxylin and eosin tissue sections


You never got rid of the mountant.  The mountant used on the tape is activated when it comes in contact with xylene. So by putting the slides back in xylene, you only reactivated it.  After you remove the coverslip, you must alternately rinse in water and dip in fresh acetone( not the one you used to take the covers. off).  The endpoint of this step is easy to see because the mountant will turn white when it comes in contact with the water.  Keep dipping and rinsing until all the white gunk is gone.  Good luck!
Juan C. Gutierrez, HT(ASCP)
Histology Supervisor
Christus Santa Rosa Healthcare
San Antonio, TX  78207

	-----Original Message----- 
	From: Peter Smale [] 
	Sent: Tue 11/25/2003 2:17 AM 
	Subject: [Histonet] restaining of hematoxylin and eosin tissue sections
	I wonder if someone can assist me in solving this problem.I retrieved a hematoxylin and eosin/phloxine stained slide from 1998 and discovered it was faded.The tissue section was mounted in a coverslipping machine that uses coverslipping tape composed of cellulose triacetate and is coated on one side with resinous mountant.This is the procedure I followed to decoverslip,decolorise and restain: I placed the slide in acetone for 7 minutes to remove the coverslip and then put the slide in xylene for 6 hours to remove any residual resinous mountant.I took the slide through graded ethanols to water after which I decolorised with acid/alcohol for one minute, took the slide back to water and restained the nuclei with Gill's Hematoxylin ( for 5 min. ) and counterstained with a mixture of 2% eosin y and 1% phloxine B with 4 drops of conc. glacial acetic acid added ( for 3 min. ).The nuclear staining turned out pale and the counter stain was patchy almost as if something was blocking the tissue's ability to retain the dyes I cannot figure out why the tissue is not staining with the same intensity as it did in 1998.Please keep in mind the slide was left out in natural light for a while which resulted in it becoming faded.
	Peter Smale
	Anatomical Pathology
	National Health Laboratory Service
	Chris Hani Baragwanath Hospital
	Soweto, Johannesburg, South Africa
	tel:   +2711 4898711
	fax:  +2711 4898717 
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