> > 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 > [email protected] > > 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 <[email protected]> > To: Histonet <[email protected]> > 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 > > > _______________________________________________ Histonet mailing list [email protected] http://lists.utsouthwestern.edu/mailman/listinfo/histonet
