Thanks for the advice! We have been worried that wax was the issue. I do
leave them in 3 sets of xylene for 5 minutes each, you would think that would
get rid of the wax. Is it ok to leave them in the xylenes for longer than 5
minutes each? Thanks again for the advice to everyone who responded so far :)
> From: tony.henw...@health.nsw.gov.au
> To: karabo...@hotmail.com; histonet@lists.utsouthwestern.edu
> Subject: RE: [Histonet] Question regarding staining of ligament tissue using
> H&E. The nuclei in our ligament tissue is not staining consistently.
> Date: Mon, 23 Jan 2012 22:38:28 +0000
>
> 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.
>
> Regards
> 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-boun...@lists.utsouthwestern.edu
> [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Kara Lee
> Sent: Tuesday, 24 January 2012 5:59 AM
> To: histonet@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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