[Histonet] Help to: Nuclear Fast Red Counterstain (Eddie Martin) Histonet Digest, Vol 261, Issue 5

[Eddie Martin]

>
> Hello Mehndi,
>
> Our lab hasn’t experienced this in some time…generally its resolved with
> adding extra online washes with surfactant post NFR counterstain. There are
> easily 3-4 practices done in a high volume histolab that may easily
> exacerbate the crystals you’re mentioning. Common practices such as:
>
> ·       letting slides dry on the special stain instrumentation
>
> ·       dehydrating too quickly after offline DI water rinses
>
> ·       Oven drying above 45C
>
> ·       The histolab’s humidity is greater than 60%
>
> Assuming the NFR commercially available reagent is properly made at an
> appropriate concentration of NFR, and your laboratory’s humidity level is
> between 40% - 60%, and the slides counterstained with NFR weren’t left to
> dry on the special stain instrument or when performed manually, then the
> following can be done to remove residual NFR crystals from embedding onto
> the slide:
>
> Note: If your Special Stain instrumentation has a slide drying process at
> the end of the run, disable it and perform the following offline steps
> while the slides are still wet after the rinsing steps on the Special Stain
> protocol:
>
>    1. Tween 20 rinse (0.05–1.0%): 1 min with gentle agitation. This will
>    remove loose precipitate left after NFR is rinsed off.
>    2. Acetic dip (optional yet highly suggested) : A few dips in 1%
>    glacial acetic acid. An acetic acid dip protonates residual dye. Any
>    residual crystals, if present on the wet slide have another opportunity to
>    be removed by charging the precipitated crystals to enable them to be
>    rinsed off the slides. Rinses in DI water is necessary to stop the acidity
>    effect.
>
> ·       If skipping acetic dip: go straight to DI water rinses after
> Tween 20 rinse.
>
>    3. Graded alcohols – Quick passes only:
>
> ·       70%: 15–30 sec
>
> ·       80%: 15 sec
>
> ·       95%: 15–30 sec
>
> ·       100%: 15–30 sec (×2)
>
> ·       Caution: Excess time in alcohol bleaches NFR.
>
> ·       Caution: Skipping graded alcohols to get to 100% alcohol sooner
> also causes precipitate to form.
>


>
>    4. Xylene: Extend time in the second xylene if the first xylene is not
>    changed frequently. (this avoids accidental bleaching in first xylene with
>    excess residual 100% alcohol).
>    5. Oven drying: if choosing to oven dry instead of dehydrating through
>    graded alcohols; Slide drying with warm temps between 40 °C   & 60 °C.
>    Slide oven temps >60 °C may also cause uneven drying and crystal
>    precipitation.
>    6. Other factors previously mentioned above: Over-concentrated NFR
>    either commercially available or when making a working solution, high
>    humidity >65%, and shortcuts taken in graded alcohols or elevating oven
>    temps above 40 °C  each individually contribute to crystals embedding onto
>    the slide.
>
>
> I hope this helps!
>
> Eddie Martin
> Technical Pathology Specialist
> The National Institutes of Health
> 10 Center Drive
> Building 10, RM 2C301
> Bethesda, MD 20816
> (301) 594-2054
> eddie.martin at nih.gov
>


> When replying, please edit your Subject line so it is more specific
> than "Re: Contents of Histonet digest..."
> Today's Topics:
>
>    1. Nuclear fast red counterstain (Histology)
>    2. Re: Nuclear fast red counterstain (John Kiernan)
>
>
>
> ---------- Forwarded message ----------
> From: Histology <histo at pathlab.us>
> To: Histonet <Histonet at lists.utsouthwestern.edu>
> Cc:
> Bcc:
> Date: Tue, 19 Aug 2025 17:31:00 +0000
> Subject: [Histonet] Nuclear fast red counterstain
> Hi all,
>
> Has anyone experienced commercially pre-made Nuclear Fast Red having
> reddish crystals in it?  We use this as a counterstain for Prussian Iron
> and Melanin stains and we are seeing little crystals on the slide.  We have
> tried filtering the solution but we are still seeing this problem.  Any
> help would be much appreciated.
>
> Thanks as always Histonet!!
>
> Mehndi Helgren
> Lab Manager
> 757-664-7901
> Dominion Pathology Labs.
> 733 Boush St. Suite 200
> Norfolk, VA  23510
>
>
>