[Histonet] Weak Hematoxylin Staining?

[Tony Henwood (SCHN)]

Vivian,

After cutting the frozen sections, how are you fixing them?

If they are air-dried then they will tend to give a washed out nuclear stain.
I would suggest immediate fixation of the sections in either methanol or to make even a brighter H&E: 1% acetic acid in ethanol (1ml glacial acetic acid + 99ml absolute ethanol), about 1 minute should suffice for both fixations.

Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 


-----Original Message-----
From: Vivian Hou via Histonet [mailto:histonet at lists.utsouthwestern.edu] 
Sent: Tuesday, 6 December 2016 7:58 AM
To: histonet at lists.utsouthwestern.edu
Subject: [Histonet] Weak Hematoxylin Staining?

Dear all,

We are doing some immunofluorescent stains that we want to pair with H&E slides. We are using fresh frozen sections for both (unfixed tissue, human diseased coronary arteries) but I am seeing very weak hematoxylin staining to the point where I have to dial back the eosin (3 min hematoxylin, 1 dip eosin).


I am using Gills 2 (newly purchased), I know its not necessary to filter it but we have been just in case using Whatman no. 5 filters. No differentiation due to the weak staining right now.


I would appreciate any thoughts on why the staining might be so weak, we do a great deal of processing and imaging on the arteries (about 12 hrs @ 25C) prior to histo and I wonder if this has cause the tissue to decay?


Thank you for your time,
V

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-----------------------------------------------------------------------------------------
V Hou
Bioengineering | Human Photonics Laboratory
e: houv2 at uw.edu | c: 206-999-3708
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