[Histonet] Question regarding staining of ligament tissue using H&E. The nuclei in our ligament tissue is not staining consistently.

Tony Henwood (SCHN) tony.henwood <@t> health.nsw.gov.au
Mon Jan 23 16:38:28 CST 2012

Have a look at the dewaxing part of the protocol. 
Is the xylene removing all the wax? 
If wax is incompletely removed from the sections then nuclei will be poorly stained whereas, interestingly the eosin counterstain will seem to be unaffected (though with a diligent look you will see poorer eosin staining as well - look at the contrast between collagen, smooth muscle and nerves).

After xylene and alcohol, check the water rinsed slide. The tissue should not be hydrophobic.

Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Laboratory Manager & Senior Scientist 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Kara Lee
Sent: Tuesday, 24 January 2012 5:59 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Question regarding staining of ligament tissue using H&E. The nuclei in our ligament tissue is not staining consistently.

Tissue is fixed in 10% NBF before being vacuum embedded in paraffin. Our tissue is cut at 10microns, placed on charged slides, then placed on a slide warmer over night.  The slides are then place in xylenes 3 times for 2 minutes, then stained as follows..Step 4:  100% Alcohol - 2 X 2 minutes each,Step 5:  95% Alcohol - 2 X 2 minutes each,Step 6:  DI H2O - 2 X 2 minutes each,Step 7:  Harris Hematoxylin - 1 X 1.5 minutes,Step 8:  Wash gently in DI H2O until"Grape Juice" color is gone, Step 9:  Acid Alcohol - 3 Dips, Step 10:  Wash gently in DI H2O - 1 X 2 minutes, Step 11:  Bluing - 10 Dips, Step 12:  Rinse in running DI H2O - 1 X 2minutes, Step 13: 95% Alcohol - 1 X 2 minutes ,Step 14:  Working Eosin - 1 X 2 minutes, Step 15: 95% Alcohol - 2 X 2 minutes each, Step 16:  100% Alcohol - 3 X 2 minutes each,Step 17: Xylene Dips - 3 X 5 minutes each, Step 18:  Coverslip.  
Our core lab has recently had a change in pressure for the DI port and water comes out very hard, making gentle washing impossible. The reagents are new.  We have tried increasing the staining time in the hematoxylin to 2 minutes and reducing the acid alcohol dips to 2. 
Our hematoxylin is not consistently staining the nuclei in the ligament tissue.  Some are good, some are bad.  
Can someone make any suggestions?
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