Hoechst 33342

Manufactured by LI COR

Hoechst 33342 is a fluorescent dye that binds to the minor groove of double-stranded DNA. It exhibits blue fluorescence upon binding to DNA and is commonly used for nucleic acid staining and cell analysis applications.

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Lab products found in correlation

Glycerol solution, by Merck Group (2 mentions) Epitope retrieval solution 1, by Leica (2 mentions) Bond dewax solution, by Leica (2 mentions) Bond rxautomated ihc ish stainer, by Leica (2 mentions)

3 protocols using hoechst 33342

Multimodal Tissue Imaging by t-CyCIF

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The t-CyCIF experimental protocol was conducted as previously
described (Du et al., 2019 (link); Lin et al., 2018 ). In brief, the mouse
and human lung adenocarcinoma and human melanoma FFPE slides were baked at
60°C for 30 min, dewaxed using Bond Dewax Solution (Leica Biosystems)
at 72°C, and antigen retrieval was performed with Epitope Retrieval 1
Solution (Leica Biosystems) at 100°C for 20 minutes using the BOND RX
Automated IHC/ISH Stainer (Leica Biosystems). All antibodies were diluted in
Odyssey Intercept Buffer (plus Hoechst 33342 0.25 μg/mL; LI-COR
Biosciences) and incubated overnight at 4°C in the dark. See the
Key Resources Table for the
complete list of antibodies. Slides were coverslipped using 20–50%
glycerol solution (Sigma-Aldrich) in PBS. Images were taken using DAPI,
FITC, Cy3, and Cy5 channels on the RareCyte CyteFinder Instrument
(20x/0.75NA objective lens). After imaging, the fluorophores were
inactivated with photobleaching solution (4.5% H2O2 and 20 mM NaOH in PBS)
for 45 minutes under LED ligfFigurehts.

Burger M.L., Cruz A.M., Crossland G.E., Gaglia G., Ritch C.C., Blatt S.E., Bhutkar A., Canner D., Kienka T., Tavana S.Z., Barandiaran A.L., Garmilla A., Schenkel J.M., Hillman M., de los Rios Kobara I., Li A., Jaeger A.M., Hwang W.L., Westcott P.M., Manos M.P., Holovatska M.M., Hodi F.S., Regev A., Santagata S, & Jacks T. (2021). Antigen Dominance Hierarchies Shape TCF1+ Progenitor CD8 T Cell Phenotypes in Tumors. Cell, 184(19), 4996-5014.e26.

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Multiplexed Tissue Imaging using t-CyCIF

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t-CyCIF was performed as described in (Lin et al., 2018; Burger et al., 2021) and at protocols.io (dx.doi.org/10.17504/protocols.io.bjiukkew). FFPE slides were baked at 60 °C for 30 min using BOND RX Automated IHC/ISH Stainer and dewaxed using Bond Dewax solution at 72 °C. Antigen retrieval was performed using Epitope Retrieval 1 (LeicaTM) solution at 100 °C for 20 min. For multiplexed imaging, each slide underwent multiple cycles of antibody incubation, imaging, and fluorophore inactivation. Antibodies along with Hoechst 33342 (0.25 μg/mL; LI-COR Biosciences) for DNA staining were diluted in Odyssey Intercept Buffer, and the slides were incubated in primary antibodies (1:100 dilution) overnight at 4 °C in the dark. Glass coverslips were wet mounted before imaging using 250 μL of 70% glycerol in 1X PBS. Images were acquired using the CyteFinder II HT Instrument, an automated slide scanning fluorescence microscope (RareCyte Inc. Seattle WA) with a 20X /0.75 NA objective. After image acquisition, slides were soaked in 42 °C PBS to facilitate coverslip removal. After slides were decoverslipped, they were incubated in a solution of 4.5% H2O2 and 20 mM NaOH in PBS under an LED light source for 45 min X 2 for fluorophore inactivation.

Zhang X., Pant S.M., Ritch C.C., Tang H.Y., Shao H., Dweep H., Gong Y.Y., Brooks R., Brafford P., Wolpaw A.J., Lee Y., Weeraratna A., Sehgal A., Herlyn M., Kossenkov A., Speicher D., Sorger P.K., Santagata S, & Dang C.V. (2024). Cell state dependent effects of Bmal1 on melanoma immunity and tumorigenicity. Nature Communications, 15, 633.

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Multiparametric Imaging of Pancreatic Cancer

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t-CyCIF imaging consisted of multiple cycles of antibody incubation, imaging, and fluorophore inactivation. The t-CyCIF experimental protocol was conducted as previously described (Du et al., 2019 (link); Lin et al., 2018 ). In brief, 5 micron sections from 9 FFPE pancreatic adenocarcinoma specimens were baked at 60°C for 30 min, dewaxed using Bond Dewax Solution (Leica Biosystems) at 72°C, and antigen retrieval was performed with Epitope Retrieval 1 Solution (Leica Biosystems) at 100°C for 20 minutes using the BOND RX Automated IHC/ISH Stainer (Leica Biosystems). All antibodies were diluted in Odyssey Intercept Buffer (plus Hoechst 33342 0.25 μg/mL; LI-COR Biosciences) and incubated overnight at 4°C in the dark. See the Key Resources Table for the complete list of antibodies (note that PDGFRα staining was non-specific and inadequate to detect iCAFs). Slides were coverslipped using 20–50% glycerol solution (Sigma-Aldrich) in PBS. Images were taken using DAPI, FITC, Cy3, and Cy5 channels on the RareCyte CyteFinder Instrument (20x/0.75NA objective lens, RareCyte Inc. Seattle WA). After imaging, the fluorophores were inactivated by incubating with photobleaching solution (4.5% H2O2 and 20 mM NaOH in PBS) for 30 minutes under LED lights (this step was repeated twice).

Koikawa K., Kibe S., Suizu F., Sekino N., Kim N., Manz T.D., Pinch B.J., Akshinthala D., Verma A., Gaglia G., Nezu Y., Ke S., Qiu C., Ohuchida K., Oda Y., Lee T.H., Wegiel B., Clohessy J.G., Sanatagata S., Wulf G.M., Hidalgo M., Muthuswamy S., Nakamura M., Gray N.S., Zhou X.Z, & Lu K.P. (2021). Targeting Pin1 Renders Pancreatic Cancer Eradicable by Synergizing with Immunochemotherapy. Cell, 184(18), 4753-4771.e27.

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