[Histonet] Weak Hematoxylin Staining?

[Vivian Hou]

Hi Tony,

I have not been fixing them but will try the 1% acetic in ethanol and the
methanol solutions, thank you for the suggestion!

Best,
Vivian

On Mon, Dec 5, 2016 at 2:52 PM, Tony Henwood (SCHN) <
tony.henwood at health.nsw.gov.au> wrote:

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