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Agreed. I’m not sure what the user is responding to but the bright nuclear regions are “chromocenters” - regions of AT-rich satellite sequences that bind strongly to Hoechst/DAPI dyes.

Confocal images of UV excitable dyes like Hoechst/DAPI tend to have low S/N. This is in part attributable to low throughput of the excitation light through the objective and/or lightguide, and may be further exacerbated by objectives with less-than-stellar chromatic correction, since both the excitation and the emission may not be focused in the proper plane and thus miss the pinholes. 

I don’t see high background in the Hoechst image you posted, but background staining with Hoechst/DAPI can be reduced by a) not including these dyes in mounting media and b) washing with buffer 1-2 times after staining, since unbound Hoecsht/DAPI does fluoresce, albeit less brightly than DNA-bound dye. At low concentrations of dye this is less of an issue. You mention that the Hoechst was diluted 1:10,000 but not its final concentration.

-Abby

> On Apr 21, 2022, at 9:54 AM, Cammer, Michael <[log in to unmask]> wrote:
> 
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> 
> I converted the image to grayscale, so I could actually assess the blue channel, and posted it at http://microscopynotes.com/temp/temp_delete_after_20220601 
> 
> The nuclei look normal.  Some are large and round.  Some are smaller and more elongated or variable morphology.  A cell at 9:00 just completed division so DNA compact in each daughter cell.  Big cells nuclei with the same total DNA as smaller nuclei may appear less bright because they are lower concentration DNA.  Anyhow, this looks normal.
> 
> The protocol does not describe permeabilzation.  Saponin or Triton?  This is critical.
> 
> Cheers-
> 
> Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory
> NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY  10016
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> 
> -----Original Message-----
> From: Confocal Microscopy List <[log in to unmask]> On Behalf Of Arvydas Matiukas
> Sent: Thursday, April 21, 2022 12:30 PM
> To: [log in to unmask]
> Subject: Re: Strange Hoechst signal
> 
> [EXTERNAL]
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> 
> Hello Microscopists,
> 
> I am seeking your advice on behalf of my Core confocal user. He is concerned about nuclear images produced by Hoechst labeling: a) some areas show no detail structure (look like saturated, though 8 bit signal levels are <200); b) high background level.
> 
> I suspect that this may be related to the sample prep protocol as other samples from the same and other labs have normal nuclear signal and the confocal is performing within specs. Our core does not provide sample prep service so labs are responsible themselves for this important step.
> 
> I attach 3 color image, acquired with 20x objective on PMT detectors and sample description.
> https://imgur.com/a/8J2Sliq
> 
> 
> Cells are oligodendrocytes progenitor cells Stained by Immunocytochemistry Pfa 4% fixed for 10 min then blocked with 30% donkey serum in PBS.
> Primary antibody over night. Fridge
> 
> 2nd ab 2 hours at room temperature
> 
> Olig2 2 goat antibody 2nd ab 598
> Pdgfralpha rabbit ab 2nd ab is 488
> Hoechst applied at 1:10000 dilution.
> 
> Any insights/comments how to improve nuclear signal are appreciated.
> 
> Best,
> Arvydas
> **********************
> Director of Microscopy Core
> SUNY Upstate Medical University
> Syracuse, NY
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