# Probes & Prep **URL:** https://forum.microlist.org/c/probes-prep/9.md [Latest](https://forum.microlist.org/latest.md) · [Categories](https://forum.microlist.org/categories.md) · [Tags](https://forum.microlist.org/tags.md) --- ## [About the Probes & Prep category](https://forum.microlist.org/t/about-the-probes-prep-category/18)
**Author:** [@talley](https://forum.microlist.org/u/talley)\ **Replies:** 0
This category is for questions about sample preparation Fluorescent proteins, organic dyes, & other labels Labeling protocols & advice If it has to do with preparing your sample prior to putting it on the microscope, … --- ## [Long-term Live Cell Imaging tips/tricks](https://forum.microlist.org/t/long-term-live-cell-imaging-tips-tricks/70)
**Author:** [@Maikel](https://forum.microlist.org/u/Maikel)\ **Replies:** 23\ **Last updated:** [September 4, 2026, 10:49am UTC](https://forum.microlist.org/t/long-term-live-cell-imaging-tips-tricks/70 "2026-09-04T10:49:32Z")
Hello – My project requires me to image cultured cells (macrophages) for up to 72 hours on an epi-fluorescence microscope. Are there any specific tips and tricks that people have that I should consider when doing these e… --- ## [Mitobrilliant stain on FFPE tissue](https://forum.microlist.org/t/mitobrilliant-stain-on-ffpe-tissue/3390)
**Author:** [@DaniilShabashvili](https://forum.microlist.org/u/DaniilShabashvili)\ **Replies:** 0\ **Last updated:** [July 2, 2026, 8:18pm UTC](https://forum.microlist.org/t/mitobrilliant-stain-on-ffpe-tissue/3390 "2026-07-02T20:18:50Z")
Has anyone tried mitobrilliant stain on FFPE tissue? We have a client who wants to do this but can’t find anything in literature. Thank you! --- ## [Staining live/dead with organoids embedded in Matrigel](https://forum.microlist.org/t/staining-live-dead-with-organoids-embedded-in-matrigel/3389)
**Author:** [@DaniilShabashvili](https://forum.microlist.org/u/DaniilShabashvili)\ **Replies:** 1\ **Last updated:** [July 2, 2026, 8:17pm UTC](https://forum.microlist.org/t/staining-live-dead-with-organoids-embedded-in-matrigel/3389 "2026-07-02T20:17:33Z")
We have a user trying to use this live-dead kit to stain cells in a layer of Matrigel, then image with a Keyence microscope LIVE/DEAD™ Viability/Cytotoxicity Kit, for mammalian cells 1 kit | Buy Online His staining does… --- ## [DAPI in plant tissue - nonspecific binding to cell walls and chloroplasts](https://forum.microlist.org/t/dapi-in-plant-tissue-nonspecific-binding-to-cell-walls-and-chloroplasts/3346)
**Author:** [@renata\_schnablova](https://forum.microlist.org/u/renata_schnablova)\ **Replies:** 0\ **Last updated:** [May 19, 2026, 6:25pm UTC](https://forum.microlist.org/t/dapi-in-plant-tissue-nonspecific-binding-to-cell-walls-and-chloroplasts/3346 "2026-05-19T18:25:28Z")
Dear all. I am facing difficulties with staining of fixed plant tissue in diverse herbaceous species. In some species (about 10% of the studied species), I see nonspecific staining of cell walls and chloroplasts, withou… --- ## [Anti-evaporation oil in live imaging: any concerns about increased hydrostatic pressure?](https://forum.microlist.org/t/anti-evaporation-oil-in-live-imaging-any-concerns-about-increased-hydrostatic-pressure/3325)
**Author:** [@cherryhuang0120](https://forum.microlist.org/u/cherryhuang0120)\ **Replies:** 0\ **Last updated:** [April 15, 2026, 6:23pm UTC](https://forum.microlist.org/t/anti-evaporation-oil-in-live-imaging-any-concerns-about-increased-hydrostatic-pressure/3325 "2026-04-15T18:23:39Z")
I recently tested the ibidi anti-evaporation oil in a cell migration assay. It worked well in maintaining the medium height—the oil-treated wells showed minimal evaporation, while the untreated controls dropped significa… --- ## [Opal 690 Bleed-Through - Has Anyone Else Seen This?](https://forum.microlist.org/t/opal-690-bleed-through-has-anyone-else-seen-this/3277)
**Author:** [@virsicas](https://forum.microlist.org/u/virsicas)\ **Replies:** 5\ **Last updated:** [March 2, 2026, 9:31am UTC](https://forum.microlist.org/t/opal-690-bleed-through-has-anyone-else-seen-this/3277 "2026-03-02T09:31:31Z")
Hello everyone, I’m hoping to get some help on an issue we’re having with Opal 690 in multiplex immunofluorescence. A user of ours is using Opal 690 for a multiplex panel on a confocal system. Even though we are using s… --- ## [Janelia Fluor Shelf Life](https://forum.microlist.org/t/janelia-fluor-shelf-life/99)
**Author:** [@MJS88](https://forum.microlist.org/u/MJS88)\ **Replies:** 11\ **Last updated:** [February 27, 2026, 6:42am UTC](https://forum.microlist.org/t/janelia-fluor-shelf-life/99 "2026-02-27T06:42:45Z")
Hello all, I recently obtained some JF549-Halo ligand dye that I’d like to use in my experiments. The Promega protocol recommends diluting in DMSO, and cautions that this dilution is single-use. Given the high cost of t… --- ## [Any anti-bleaching strategies for cells mounted with PBS?](https://forum.microlist.org/t/any-anti-bleaching-strategies-for-cells-mounted-with-pbs/3308)
**Author:** [@Topaz2026](https://forum.microlist.org/u/Topaz2026)\ **Replies:** 2\ **Last updated:** [February 17, 2026, 8:38am UTC](https://forum.microlist.org/t/any-anti-bleaching-strategies-for-cells-mounted-with-pbs/3308 "2026-02-17T08:38:52Z")
Hi everyone, I am now using water objective imaging samples in PBS, which provided no RI mismatch. But the sample in PBS was easier to bleach without any anti-bleaching agents added. I would like to change PBS to mounti… --- ## [Methods for more uniform FP yeast expression?](https://forum.microlist.org/t/methods-for-more-uniform-fp-yeast-expression/3280)
**Author:** [@ColinD](https://forum.microlist.org/u/ColinD)\ **Replies:** 1\ **Last updated:** [December 1, 2025, 3:58pm UTC](https://forum.microlist.org/t/methods-for-more-uniform-fp-yeast-expression/3280 "2025-12-01T15:58:45Z")
I have different strains of S.c yeast expressing equimolar amounts of eGFP amd mCherry from a resident plasmid (pDRF1-GW-eGFP-T2A-mCherry). When a cell expresses GFP it also, as expected, expresses mCherry. However, many… --- ## [Difference between TetraSpeck beads slides and TetraSpeck beads in suspension](https://forum.microlist.org/t/difference-between-tetraspeck-beads-slides-and-tetraspeck-beads-in-suspension/3242)
**Author:** [@esther.raeth](https://forum.microlist.org/u/esther.raeth)\ **Replies:** 3\ **Last updated:** [October 22, 2025, 3:21pm UTC](https://forum.microlist.org/t/difference-between-tetraspeck-beads-slides-and-tetraspeck-beads-in-suspension/3242 "2025-10-22T15:21:51Z")
Hello, I encountered differences in the PSF FWHM between pre-mounted commercial TetraSpeck beads slides and self-made custom beads slides using TetraSpeck beads in suspension (same actual bead diameter / microscope / PS… --- ## [Dual fluorophore expression in mammalian systems](https://forum.microlist.org/t/dual-fluorophore-expression-in-mammalian-systems/3211)
**Author:** [@Katherineimages](https://forum.microlist.org/u/Katherineimages)\ **Replies:** 6\ **Last updated:** [September 11, 2025, 10:07am UTC](https://forum.microlist.org/t/dual-fluorophore-expression-in-mammalian-systems/3211 "2025-09-11T10:07:30Z")
Hello! I am doing a lot of live mammalian cell imaging with fluorescent proteins but I am struggling to express two fluorescent proteins in my system. One of which is an inducible FP and so I initially tried to optimize… --- ## [FLIM: Easy sample for product demonstrations?](https://forum.microlist.org/t/flim-easy-sample-for-product-demonstrations/3172)
**Author:** [@phoTONIcs](https://forum.microlist.org/u/phoTONIcs)\ **Replies:** 3\ **Last updated:** [July 24, 2025, 8:09am UTC](https://forum.microlist.org/t/flim-easy-sample-for-product-demonstrations/3172 "2025-07-24T08:09:24Z")
Hello Micro-community! Is there an “easy to prep” (non-labor intensive/few reagents) FLIM sample to use to train microscopy users? Thats visually impressive, but gets the job done? --- ## [What are the main causes of poor axial resolution in light sheet microscopy? (SPIM)](https://forum.microlist.org/t/what-are-the-main-causes-of-poor-axial-resolution-in-light-sheet-microscopy-spim/3151)
**Author:** [@engpol](https://forum.microlist.org/u/engpol)\ **Replies:** 5\ **Last updated:** [July 10, 2025, 12:29pm UTC](https://forum.microlist.org/t/what-are-the-main-causes-of-poor-axial-resolution-in-light-sheet-microscopy-spim/3151 "2025-07-10T12:29:52Z")
First post on here, apologies beforehand for any mistakes in etiquette. I should also say that I am very new to the field of light sheet microscopy, so please do correct me if anything I say is misguided. P.S Apparently… --- ## [NIR Spectra Beads](https://forum.microlist.org/t/nir-spectra-beads/3107)
**Author:** [@edelaserna](https://forum.microlist.org/u/edelaserna)\ **Replies:** 0\ **Last updated:** [May 9, 2025, 3:06pm UTC](https://forum.microlist.org/t/nir-spectra-beads/3107 "2025-05-09T15:06:50Z")
Hi µforum! I am curious if anyone has a favorite near infrared (e.g., similar spectra to Cy7 or AlexaFluor 750) fluorescent bead option for imaging on the various point-scanning confocal setups that have the NIR laser/d… --- ## [405-nm excitation conjugated phalloidin](https://forum.microlist.org/t/405-nm-excitation-conjugated-phalloidin/1788)
**Author:** [@cleterrier](https://forum.microlist.org/u/cleterrier)\ **Replies:** 1\ **Last updated:** [February 20, 2025, 11:10am UTC](https://forum.microlist.org/t/405-nm-excitation-conjugated-phalloidin/1788 "2025-02-20T11:10:50Z")
SIM/confocal folks, what’s the latest on usable fluorescent phalloidin with a 405 nm laser line? Our preferred option (DyLight 405) is not available. AF405+? Atto something? CF405M? iFluor 405? I don’t think Brilliant Vi… --- ## [Stickyness of fluorophores: Thiol reactivity and xanthene derivative dyes?](https://forum.microlist.org/t/stickyness-of-fluorophores-thiol-reactivity-and-xanthene-derivative-dyes/2920)
**Author:** [@Daniel\_Furth](https://forum.microlist.org/u/Daniel_Furth)\ **Replies:** 2\ **Last updated:** [December 16, 2024, 4:33pm UTC](https://forum.microlist.org/t/stickyness-of-fluorophores-thiol-reactivity-and-xanthene-derivative-dyes/2920 "2024-12-16T16:33:49Z")
We have started to get some issues that we didn’t have before and its getting quite annoying so wanted to check if someone have experience with this before. HeLa cells where we have previously used AZdye594-DBCO to labe… --- ## [Hardening mounting media that doesn't compress sample?](https://forum.microlist.org/t/hardening-mounting-media-that-doesnt-compress-sample/416)
**Author:** [@LPUoO](https://forum.microlist.org/u/LPUoO)\ **Replies:** 20\ **Last updated:** [February 14, 2023, 4:47pm UTC](https://forum.microlist.org/t/hardening-mounting-media-that-doesnt-compress-sample/416 "2023-02-14T16:47:03Z")
Hi, Are there hardening mounting media that really don’t compress the sample when hardening ? Below are images of the same cells mounted with Vectashield, or Prolong Gold/Glass and only the cells in Vectashield look th… --- ## [Imaging isolated live red blood cells / erythrocytes](https://forum.microlist.org/t/imaging-isolated-live-red-blood-cells-erythrocytes/1655)
**Author:** [@innagol](https://forum.microlist.org/u/innagol)\ **Replies:** 3\ **Last updated:** [October 25, 2024, 10:04am UTC](https://forum.microlist.org/t/imaging-isolated-live-red-blood-cells-erythrocytes/1655 "2024-10-25T10:04:36Z")
Hi everyone, We are trying to image isolated live RBC on a glass-bottom dish in a WF incubated system. Unfortunately, we have problems like that after several hours, we see round accumulations in the RBC that are fluor… --- ## [Mounting medium for multiphoton microscopy](https://forum.microlist.org/t/mounting-medium-for-multiphoton-microscopy/491)
**Author:** [@Alex.h](https://forum.microlist.org/u/Alex.h)\ **Replies:** 5\ **Last updated:** [October 23, 2024, 8:35am UTC](https://forum.microlist.org/t/mounting-medium-for-multiphoton-microscopy/491 "2024-10-23T08:35:07Z")
Hello, We did some experiment with mounting medium and some mounting medium “burn” the slice sample (I see some sort of black spot on the slice and more i stay without movement more they become bigger) during the acquis… --- ## [Is SlowFade Glass supposed to look brown?](https://forum.microlist.org/t/is-slowfade-glass-supposed-to-look-brown/2557)
**Author:** [@JelleP](https://forum.microlist.org/u/JelleP)\ **Replies:** 3\ **Last updated:** [June 8, 2024, 3:07am UTC](https://forum.microlist.org/t/is-slowfade-glass-supposed-to-look-brown/2557 "2024-06-08T03:07:23Z")
Hi all, I’ve decided to give SlowFade a try. A couple weeks ago I purchased a bottle, and mistakenly stored it above the recommended temperature (4 degrees, instead of \< -5 degrees…) Upon the first time opening it (to… --- ## [Mounting media RI](https://forum.microlist.org/t/mounting-media-ri/2535)
**Author:** [@szh141](https://forum.microlist.org/u/szh141)\ **Replies:** 10\ **Last updated:** [May 30, 2024, 8:09pm UTC](https://forum.microlist.org/t/mounting-media-ri/2535 "2024-05-30T20:09:52Z")
Hello everyone. I have one question about one particular mounting media, Immuno Mount Epredia Immu-Mount:Histology and Cytology:Slide Mounting Media | Fisher Scientific, our user used to mount the sample. In the descript… --- ## [Staining edge effect with Akoya Phenocycler Fusion but not IHC](https://forum.microlist.org/t/staining-edge-effect-with-akoya-phenocycler-fusion-but-not-ihc/2317)
**Author:** [@wflynny](https://forum.microlist.org/u/wflynny)\ **Replies:** 3\ **Last updated:** [April 16, 2024, 3:44pm UTC](https://forum.microlist.org/t/staining-edge-effect-with-akoya-phenocycler-fusion-but-not-ihc/2317 "2024-04-16T15:44:18Z")
Hello, wondering if anyone may have suggestions to a problem we’re having with our PhenoCycler-Fusion (CODEX) from Akoya Biosciences. We’ve been running it well for several months, but recently we’ve run into issues whe… --- ## [Whole-mount muscle organoid in PDMS device - Mounting and confocal setup advice](https://forum.microlist.org/t/whole-mount-muscle-organoid-in-pdms-device-mounting-and-confocal-setup-advice/2441)
**Author:** [@Intersystem](https://forum.microlist.org/u/Intersystem)\ **Replies:** 2\ **Last updated:** [March 7, 2024, 10:00am UTC](https://forum.microlist.org/t/whole-mount-muscle-organoid-in-pdms-device-mounting-and-confocal-setup-advice/2441 "2024-03-07T10:00:36Z")
Hi all, I’m looking for some advice to optimize my experiment. I have a muscle orgnanoid tissue fixed (4% PFA) in a organ-on-chip device that I want to perform whole-mount immunostaining on in combination with confocal … --- ## [Search for AbberiorFlip565](https://forum.microlist.org/t/search-for-abberiorflip565/2337)
**Author:** [@HannahV](https://forum.microlist.org/u/HannahV)\ **Replies:** 0\ **Last updated:** [November 30, 2023, 8:57am UTC](https://forum.microlist.org/t/search-for-abberiorflip565/2337 "2023-11-30T08:57:50Z")
Hello, I’m searching for AbberiorFlip565 dye, but when I contacted Aberrior they told me that this series was discontinued. Would anyone know where I could potentially get it? Or would anyone be willing to share it/sel… --- ## [Fluorescent nanobodies that target GFP?](https://forum.microlist.org/t/fluorescent-nanobodies-that-target-gfp/2330)
**Author:** [@Angus\_Rae](https://forum.microlist.org/u/Angus_Rae)\ **Replies:** 1\ **Last updated:** [November 29, 2023, 10:27am UTC](https://forum.microlist.org/t/fluorescent-nanobodies-that-target-gfp/2330 "2023-11-29T10:27:26Z")
Hi all, I’m looking for anti GFP fluorescent nanobodies conjugated with Alexa 647 (or equivalent) for immunostaining of samples with GFP in order to do dSTORM imaging. Does anyone know of any commercially available fluor… --- ## [Dyes/secondary antibodies for 2P imaging](https://forum.microlist.org/t/dyes-secondary-antibodies-for-2p-imaging/2321)
**Author:** [@lgelman](https://forum.microlist.org/u/lgelman)\ **Replies:** 2\ **Last updated:** [November 23, 2023, 1:15pm UTC](https://forum.microlist.org/t/dyes-secondary-antibodies-for-2p-imaging/2321 "2023-11-23T13:15:31Z")
Hi everybody, We are interested in dyes that perform well with 2P imaging. We have large structures stained with primary Ab and secondary Ab conjugated to fluorophores. Alexa488 works fine for us, but we would like to e… --- ## [Is it possible to re-slice cleared samples?](https://forum.microlist.org/t/is-it-possible-to-re-slice-cleared-samples/2217)
**Author:** [@J\_Ramirez](https://forum.microlist.org/u/J_Ramirez)\ **Replies:** 4\ **Last updated:** [August 30, 2023, 10:03am UTC](https://forum.microlist.org/t/is-it-possible-to-re-slice-cleared-samples/2217 "2023-08-30T10:03:21Z")
Hi there everyone, I was thinking about re-slicing samples that have undergone clearing through the DISCO method (vDISCO) in order to image them in a confocal system. Ideally, we would like to register the confocal aqui… --- ## [Alternattive to tetraspeck beads in lightsheet](https://forum.microlist.org/t/alternattive-to-tetraspeck-beads-in-lightsheet/2232)
**Author:** [@jbogomolovas2](https://forum.microlist.org/u/jbogomolovas2)\ **Replies:** 1\ **Last updated:** [August 4, 2023, 2:34pm UTC](https://forum.microlist.org/t/alternattive-to-tetraspeck-beads-in-lightsheet/2232 "2023-08-04T14:34:28Z")
LHello Everyone, I am currently using fluorescent tetraspeck beads for image registration in lightsheet multiangle microscopy. However, I’ve noticed that these beads display a notably weak signal in the DAPI channel, w… --- ## [Suitable fluorophore combinations for Tausense lifetime-based dye separation](https://forum.microlist.org/t/suitable-fluorophore-combinations-for-tausense-lifetime-based-dye-separation/1589)
**Author:** [@huwnaylor](https://forum.microlist.org/u/huwnaylor)\ **Replies:** 3\ **Last updated:** [July 24, 2023, 12:49pm UTC](https://forum.microlist.org/t/suitable-fluorophore-combinations-for-tausense-lifetime-based-dye-separation/1589 "2023-07-24T12:49:16Z")
Hello, I wondered if anyone would be able to share fluorophore combinations that are appropriate for making the most of the Tausense module in multi-channel immunofluorescence experiments for imaging on a (new!) Leica S… [Next page](https://forum.microlist.org/c/probes-prep/9.md?page=1)