, DAPI Counterstain Solution: Since DAPI passes through an intact cell membrane, it can be used to stain live cells and fixed cells. DAPI stains There are several methods that are used for fluorescent staining of actin filaments in tissue culture cells. Below we provide two protocols for staining live cells with DAPI or Hoechst. Embed the tissue in paraffin at 58 °C. //2007-09-15: IHC-Protocol-Other-Side fluorescent techniques. cytoplasmic labeling. Therefore, this will not be suitable for all antibodies, as the protein cross-linking formed by the fixative may block access of the antibody to the epitope. Its blue fluorescence google_color_border = "FFFFFF"; In S. cerevisiae, DAPI and Hoechst preferentially stain dead cells with nuclear and cytoplasmic localization. To preserve tissue morphology and retain the antigenicity of the target molecules, fix the tissue by vascular perfusion with 500 - 700 mL of Formaldehyde Fixative Solution. google_color_border = "A9A9A9"; 100 ml Deparaffinize and rehydrate by immersing the slides through the following solutions/wells: 1.1. DAPI Stock Solution (5mg/ml or 14.3 2. Take standard confocal images (at least fife frames for each sample). 1. google_color_url = "215670"; Add DAPI Dilute 1 : 20-35,000 in PBS Incubate for 10 minutes in the wet chamber Wash 2 x 3 minutes in TBS-T Wash 1 x in MQ water to remove debris from slide, dry around tissue. Counterstain for Add a drop of mounting media onto the tissue. The following protocols can be modified for tissue staining or for staining unfixed cells or tissues. DAPI and Hoechst are minor-groove binding dyes; DAPI has higher affinity for A/T-rich regions of DNA than G/C-rich DNA. DAPI (Molecular Probes, Cat# D-1306) The fixation procedure is critical for obtaining faithful representation of the F-actin distribution within the cell. 3. Fluorescence spectral characteristics. google_ad_format = "468x15_0ads_al"; Interested in Ordering? It is not recommended to store working solutions of Hoechst dye, because the dye will be lost to precipitation or adsorption to the container over time. Incubate sections in dark for 30 Fluorescent staining of frozen sections • Take slides with sections out from freezer and thaw at room temperature for 10-20 minutes . achieve stronger staining, use 4 ul stock solution or reduce PBS google_color_url = "215670"; Dilute the DAPI solution to an appropriate concentration of working solution as the manual instruction. nuclei specifically, with little or no   DAPI staining is done after staining for other markers. tissue sections or -------------------------------------- 2 ul, PBS -------------------------------------------------------- They are typically used for staining at 1 ug/mL. google_color_url = "215670"; DAPI can bind to RNA, but its selectivity for DNA over RNA is high.It appears that DAPI uses an intercalating binding mode with RNA that is AU selective (Biochem., 31, 3103 (1992)). fluorescent techniques. Mount slides with 50 - 100% glycerol and a no. Immerse the tissue in xylene (mixed isomers) three times for 20 minutes each at room temperature. Whatever fixative you have successfully used in IHC-Fr with your antibody should also be suitable for whole mount. google_ad_format = "300x250_as"; DAPI Staining Solution (ab228549) is a fluorescent stain for labeling DNA in fluorescence microscopy. Hoechst 33528 is slightly more water soluble than Hoechst 33342, but both dyes are highly cell membrane permeant and widely used in cell cycle studies and as nuclear counterstains for live or fixed cells. google_color_border = "FFFFFF"; Tip:Before moving to alcohol grades step, make sure to completely deparaffinize the sections. Staining by medium exchange results in uniform exposure of cells to probe. PBS -------------------------------------------------------- 300 nM in PBS): in multicolor stands out in vivid contrast to green, Its blue fluorescence For example, did you know photoconversion of DAPI can cause nuclear stains to fluoresce? Incubate at room temperature for 5 min. Antifade mounting medium: Fluoromount G (refractive index 1.393; Southern Biotech, cat. mM): Advertise | The excitation maximum for DAPI bound to dsDNA is 358 nm, and the emission maximum is 461 nm. (DAPI) is possible. Materials. 7. How to Stain Bacteria or Yeast In comparing Hoechst staining to DAPI staining, there are a number of important things you should be aware of. All Rights Reserved. for staining cultured cells on slides. Note that we do not recommend adding highly concentrated dye directly to cells in culture, as this will result in local areas of high dye exposure. Privacy | cytoplasmic labeling. Disclaimer | Do not allow the cells to dry out at any time during the protocol. amount to 50 ml). Leave the cells in ethanol at –20°C for 5–15 minutes. Incubate cells at room temperature or 37°C for 5-15 minutes, then image. in multicolor The following protocol is used for staining of tissue sections or for staining cultured cells on slides. Protein X-red, Protein Y-green) and always stain nuclei with DAPI or Hoechst 33342. The 5 mg/mL DAPI stock solution may be stored at 2–6°C for up to 6 months or at ≤–20°C for longer periods. 100% Ethanol, two washes 10 minutes each 1.3. Remove culture medium from the cells and replace with medium containing dye. How to Stain Live Cells google_ad_type = "text_image"; The presented staining method can be a single, easy-to-use alternative for a range of other staining protocols commonly used for microscopic analyses in plant reproductive biology. no. Cells that have been immunolabeled can be stained with DAPI by starting at Step 7.   100 ml. 2. Xylene, three washes 5 minutes each 1.2. Add 2 mL of deionized water (diH 2 O) or dimethylformamide (DMF) to the entire contents of the DAPI vial to make a 14.3 mM (5 mg/mL) DAPI stock solution. 1. google_ad_height = 250; Dimethylformamide (DMF) google_color_bg = "FFFFFF"; Abnormal lipid droplet accumulation can be associated with various metabolic Nuclei will be stained google_ad_width = 160; 2.4 Transfer the full volume of resuspended cells to 4 mL of absolute ethanol at –20°C by pipetting the cell suspension slowly into the ethanol while vortexing at top speed. Place dry coverslip onto mounting media. Certificate No. DAPI Stock Solution (5mg/ml or 14.3 mM): DAPI (Molecular Probes, Cat# D-1306) ----- 10 mg. Dimethylformamide (DMF) ----- 2 ml. Incubate sections in dark for 30 50% Ethanol, two washe… google_ad_channel = "7491275008"; … Especially when it comes to photoconversion. DAPI (4′,6-Diamidino-2-Phenylindole) Staining Protocol. Learn more about the other cellular stains available to you. For example, in the image of the tumor stained with F4/80, cell surface staining of this antigen is observed. google_color_bg = "A9A9A9"; DAPI stains mM): ---------------- 10 mg The dyes can be used to stain yeast at 12-15 ug/mL in PBS. marker is used. US3564. //-->, Home | , BNCAP1595-500, BNCA1595-250, BNCB1595-100, BNCB1595-500, BNC061595-100, BNC061595-500, BNC051595-100, BNC051595-500, BNC041595-100, BNC041595-500, BNC881595-100, BNC881595-500, BNC141595-100, BNC141595-500, BNC431595-100, BNC431595-500, BNC551595-100, BNC551595-500, BNC681595-100, BNC681595-500, BNC941595-100, BNC941595-500, BNC401595-100, BNC401595-500, BNC471595-100, BNC471595-500, BNC601595-100, BNC601595-500, BNC611595-100, BNC611595-500, BNC801595-100, BNC801595-500, BNC811595-100, BNC811595-500, BNC001595-100, BNC001595-500, BNC701595-100, BNC701595-500, BNCH1595-100, BNCH1595-500, BNCP1595-250, BNUM1595-50, BNUB1595-100, BNUB1595-500. Aliquot and store in –20 ºC. ------------------------------ 2 ml. , BNCAP0074-500, BNCA0074-250, BNCB0074-100, BNCB0074-500, BNC060074-100, BNC060074-500, BNC050074-100, BNC050074-500, BNC040074-100, BNC040074-500, BNC880074-100, BNC880074-500, BNC140074-100, BNC140074-500, BNC430074-100, BNC430074-500, BNC550074-100, BNC550074-500, BNC680074-100, BNC680074-500, BNC940074-100, BNC940074-500, BNC400074-100, BNC400074-500, BNC470074-100, BNC470074-500, BNC600074-100, BNC600074-500, BNC610074-100, BNC610074-500, BNC800074-100, BNC800074-500, BNC810074-100, BNC810074-500, BNC700074-100, BNC700074-500, BNC000074-100, BNC000074-500, BNCH0074-100, BNCH0074-500, BNCP0074-250, BNUM0074-50, BNUB0074-100, BNUB0074-500, Fluorescent stains for cell surface, nucleus, mitochondria, lysosomes, & more. 8076.2) in 1 × MTSB; Calcofluor white (BR 28, Sigma, cat. The RedDot™ dyes are used to dye the nuclei of live cells, and they come in RedDot™ 1 and RedDot™ 2 options. Add the dye to complete culture medium. The following protocol is used for staining of //-->,

//-->. How to Stain Fixed Cells or Tissue Sections google_ad_type = "text_image"; google_color_bg = "FFFFFF"; DAPI is a popular nuclear counterstain for use , BNCAP2214-500, BNCA2214-250, BNCB2214-100, BNCB2214-500, BNC062214-100, BNC062214-500, BNC052214-100, BNC052214-500, BNC042214-100, BNC042214-500, BNC882214-100, BNC882214-500, BNC142214-100, BNC142214-500, BNC432214-100, BNC432214-500, BNC552214-100, BNC552214-500, BNC682214-100, BNC682214-500, BNC942214-100, BNC942214-500, BNC402214-100, BNC402214-500, BNC472214-100, BNC472214-500, BNC602214-100, BNC602214-500, BNC612214-100, BNC612214-500, BNC802214-100, BNC802214-500, BNC812214-100, BNC812214-500, BNC002214-100, BNC002214-500, BNC702214-100, BNC702214-500, BNCH2214-100, BNCH2214-500, BNCP2214-250, BNUM2214-50, BNUB2214-100, BNUB2214-500. DAPI Stock Solution (5mg/ml or 14.3 Without removing the medium from the cells, add 1/10 volume of 10X dye directly to the well. And you’ll also get access to one of our expert scientists to help you choose the best products for your research. bottle or wrapped with aluminum foil to protect from light (to Learn more tech tips just like this on our Tech Tips page. It is the protocol that I used (DAPI staining in paraffin embedded tissue) and my figures is available in my papers. We offer DAPI dilactate, a more soluble DAPI salt, which is useful for making stock solutions for cell staining, as well as ready-to-use stock solutions of DAPI and Hoechst in water (see ordering information).   Mix to dissolve and it may take sometime to completely dissolve. Note: DAPI has poor solubility in water, so sonicate as necessary to dissolve. in multicolor, ------------------------------------------------------- The DAPI Staining Solution is a ready-to-use reagent suitable for the exclusion of dead and apoptotic cells from flow cytometric analysis. //2007-09-15: IHC-Protocol-Other-Bottom google_ad_height = 600; google_ad_client = "pub-7080753133094481"; DAPI is used as a nuclear counterstain in our FISH protocol at 2 μg/mL in antifade mounting media. google_color_link = "003366"; Add the PBS with dye to cells or tissue sections and incubate at room temperature for at least 5 minutes. google_color_link = "003366"; Protocol 1 - Fixing and staining tissue culture cells with fluorescent phalloidin (F-actin), Dapi (DNA) and an antibody. Solution1*: 5 μL DAPI (10 mg/ml solution) DAPI stock solution sometime to completely dissolve. DAPI Working Solution (100ng/ml or //2007-09-15: IHC-Protocol-Other-Top Store this solution at 4 ºC in brown Wash twice for ten minutes in TBST at room temperature. minutes at room temperature. (2) ... DAPI stain: add 0.1 µg/ml DAPI in 1X PBS and incubate for 10-15 min at RT (12)Wash 3 x 5 min in 1 x PBS at RT. ---------------- 10 mg, Dimethylformamide (DMF) ------------------------------ 2 ml google_color_text = "000000"; All payment in US dollars must be payable on a US bank. minutes at room temperature. The data from the imaging will provide information regarding the intensity and localization of the protein within the tissue section. no. Mix-n-Stain™ CF® Dye Antibody Labeling Kits, TrueBlack® Lipofuscin Autofluorescence Quencher, Easy and Fun Science Experiments to Try with Kids at Home, Background Reducers for Improved Fluorescent Stains, Getting the Whole Picture with Spectral Flow Cytometry, Blowing Up the Microscopic with Expansion Microscopy, Pushing Biology Forward with Super Resolution, 20% Off Live-or-Dye ® Fixable Viability Staining Kits, Biotium implements a Quality System, certified by QAS according to Standard QAS ISO 9001:2015. DAPI Counterstain Solution: Since DAPI-containing mounting media was used, nuclei are shown in blue. Here’s our general protocol for IF staining of cells for Microscopy: Protocol: Immunofluorescence Staining of Cells for Microscopy. 1. google_color_text = "000000"; for staining cultured cells on slides. tissue sections or -------------------------------------- 2 ul Dead cells tend to stain more brightly than live cells. Counter stain with DAPI or nuclear stain of choice diluted in TBST. λ Ex /λ Em (with DNA) = 358/461 nm; MW = 457.49 (in H 2 O); 350.25 (dihydrochloride salt); 457.49 (dilactate salt) Molecular formula: C 16 H 17 Cl 2 N 5; CAS number: 28718-90-3; Recommended staining concentration: 10 ug/mL (live cells) or 1 ug/mL (fixed cells) DAPI, dihydrochloride Hoechst 33258 Staining Protocol 100 ml. DAPI (0.25 µg/ml) in H 2O (two absorption maxima: 349 nm and 263 nm) or 2. amount to 50 ml). nuclei specifically, with little or no Staining conditions for specific antibody must be optimized according to different antigens of … google_ad_format = "160x600_as"; Follow processing schedule recommended in section C. B. Fixation of Tissues in Zinc Fixative: 0100-01) or ProlongGold (refractive index 1.47; http://products.invitrogen.com/ivgn/product/P36930); Blocking solution: 2 % albumin fraction V BSA (Carl Roth, cat. DAPI stains The dyes have minimal fluorescence in solution, but become brightly fluorescent upon binding to DNA. achieve stronger staining, use 4 ul stock solution or reduce PBS Compare applications, fixability, cellular targets, and staining in different organisms at a glance. Recommended staining concentration: 10 ug/mL (live cells) or 1 ug/mL (fixed cells), Recommended staining concentration: 1 ug/mL. tissue sections or Fix cells using method of choice. Stain for a single protein or carry out double immunofluorescence combining suitable secondary antibodies (e.g. Wash slides with PBS for three times, each for 5 minutes. We're currently trying to stain 50um (or thicker) sections of mounted FFPE brain tissue, with no luck so far. About | Staining Pattern Immunohistochemistry Fixing and Sectioning Frozen Tissue Protocol. , DAPI Counterstain Solution: Since DAPI passes through an intact cell membrane, it can be used to stain live cells and fixed cells. DAPI stains There are several methods that are used for fluorescent staining of actin filaments in tissue culture cells. Below we provide two protocols for staining live cells with DAPI or Hoechst. Embed the tissue in paraffin at 58 °C. //2007-09-15: IHC-Protocol-Other-Side fluorescent techniques. cytoplasmic labeling. Therefore, this will not be suitable for all antibodies, as the protein cross-linking formed by the fixative may block access of the antibody to the epitope. Its blue fluorescence google_color_border = "FFFFFF"; In S. cerevisiae, DAPI and Hoechst preferentially stain dead cells with nuclear and cytoplasmic localization. To preserve tissue morphology and retain the antigenicity of the target molecules, fix the tissue by vascular perfusion with 500 - 700 mL of Formaldehyde Fixative Solution. google_color_border = "A9A9A9"; 100 ml Deparaffinize and rehydrate by immersing the slides through the following solutions/wells: 1.1. DAPI Stock Solution (5mg/ml or 14.3 2. Take standard confocal images (at least fife frames for each sample). 1. google_color_url = "215670"; Add DAPI Dilute 1 : 20-35,000 in PBS Incubate for 10 minutes in the wet chamber Wash 2 x 3 minutes in TBS-T Wash 1 x in MQ water to remove debris from slide, dry around tissue. Counterstain for Add a drop of mounting media onto the tissue. The following protocols can be modified for tissue staining or for staining unfixed cells or tissues. DAPI and Hoechst are minor-groove binding dyes; DAPI has higher affinity for A/T-rich regions of DNA than G/C-rich DNA. DAPI (Molecular Probes, Cat# D-1306) The fixation procedure is critical for obtaining faithful representation of the F-actin distribution within the cell. 3. Fluorescence spectral characteristics. google_ad_format = "468x15_0ads_al"; Interested in Ordering? It is not recommended to store working solutions of Hoechst dye, because the dye will be lost to precipitation or adsorption to the container over time. Incubate sections in dark for 30 Fluorescent staining of frozen sections • Take slides with sections out from freezer and thaw at room temperature for 10-20 minutes . achieve stronger staining, use 4 ul stock solution or reduce PBS google_color_url = "215670"; Dilute the DAPI solution to an appropriate concentration of working solution as the manual instruction. nuclei specifically, with little or no   DAPI staining is done after staining for other markers. tissue sections or -------------------------------------- 2 ul, PBS -------------------------------------------------------- They are typically used for staining at 1 ug/mL. google_color_url = "215670"; DAPI can bind to RNA, but its selectivity for DNA over RNA is high.It appears that DAPI uses an intercalating binding mode with RNA that is AU selective (Biochem., 31, 3103 (1992)). fluorescent techniques. Mount slides with 50 - 100% glycerol and a no. Immerse the tissue in xylene (mixed isomers) three times for 20 minutes each at room temperature. Whatever fixative you have successfully used in IHC-Fr with your antibody should also be suitable for whole mount. google_ad_format = "300x250_as"; DAPI Staining Solution (ab228549) is a fluorescent stain for labeling DNA in fluorescence microscopy. Hoechst 33528 is slightly more water soluble than Hoechst 33342, but both dyes are highly cell membrane permeant and widely used in cell cycle studies and as nuclear counterstains for live or fixed cells. google_color_border = "FFFFFF"; Tip:Before moving to alcohol grades step, make sure to completely deparaffinize the sections. Staining by medium exchange results in uniform exposure of cells to probe. PBS -------------------------------------------------------- 300 nM in PBS): in multicolor stands out in vivid contrast to green, Its blue fluorescence For example, did you know photoconversion of DAPI can cause nuclear stains to fluoresce? Incubate at room temperature for 5 min. Antifade mounting medium: Fluoromount G (refractive index 1.393; Southern Biotech, cat. mM): Advertise | The excitation maximum for DAPI bound to dsDNA is 358 nm, and the emission maximum is 461 nm. (DAPI) is possible. Materials. 7. How to Stain Bacteria or Yeast In comparing Hoechst staining to DAPI staining, there are a number of important things you should be aware of. All Rights Reserved. for staining cultured cells on slides. Note that we do not recommend adding highly concentrated dye directly to cells in culture, as this will result in local areas of high dye exposure. Privacy | cytoplasmic labeling. Disclaimer | Do not allow the cells to dry out at any time during the protocol. amount to 50 ml). Leave the cells in ethanol at –20°C for 5–15 minutes. Incubate cells at room temperature or 37°C for 5-15 minutes, then image. in multicolor The following protocol is used for staining of tissue sections or for staining cultured cells on slides. Protein X-red, Protein Y-green) and always stain nuclei with DAPI or Hoechst 33342. The 5 mg/mL DAPI stock solution may be stored at 2–6°C for up to 6 months or at ≤–20°C for longer periods. 100% Ethanol, two washes 10 minutes each 1.3. Remove culture medium from the cells and replace with medium containing dye. How to Stain Live Cells google_ad_type = "text_image"; The presented staining method can be a single, easy-to-use alternative for a range of other staining protocols commonly used for microscopic analyses in plant reproductive biology. no. Cells that have been immunolabeled can be stained with DAPI by starting at Step 7.   100 ml. 2. Xylene, three washes 5 minutes each 1.2. Add 2 mL of deionized water (diH 2 O) or dimethylformamide (DMF) to the entire contents of the DAPI vial to make a 14.3 mM (5 mg/mL) DAPI stock solution. 1. google_ad_height = 250; Dimethylformamide (DMF) google_color_bg = "FFFFFF"; Abnormal lipid droplet accumulation can be associated with various metabolic Nuclei will be stained google_ad_width = 160; 2.4 Transfer the full volume of resuspended cells to 4 mL of absolute ethanol at –20°C by pipetting the cell suspension slowly into the ethanol while vortexing at top speed. Place dry coverslip onto mounting media. Certificate No. DAPI Stock Solution (5mg/ml or 14.3 mM): DAPI (Molecular Probes, Cat# D-1306) ----- 10 mg. Dimethylformamide (DMF) ----- 2 ml. Incubate sections in dark for 30 50% Ethanol, two washe… google_ad_channel = "7491275008"; … Especially when it comes to photoconversion. DAPI (4′,6-Diamidino-2-Phenylindole) Staining Protocol. Learn more about the other cellular stains available to you. For example, in the image of the tumor stained with F4/80, cell surface staining of this antigen is observed. google_color_bg = "A9A9A9"; DAPI stains mM): ---------------- 10 mg The dyes can be used to stain yeast at 12-15 ug/mL in PBS. marker is used. US3564. //-->, Home | , BNCAP1595-500, BNCA1595-250, BNCB1595-100, BNCB1595-500, BNC061595-100, BNC061595-500, BNC051595-100, BNC051595-500, BNC041595-100, BNC041595-500, BNC881595-100, BNC881595-500, BNC141595-100, BNC141595-500, BNC431595-100, BNC431595-500, BNC551595-100, BNC551595-500, BNC681595-100, BNC681595-500, BNC941595-100, BNC941595-500, BNC401595-100, BNC401595-500, BNC471595-100, BNC471595-500, BNC601595-100, BNC601595-500, BNC611595-100, BNC611595-500, BNC801595-100, BNC801595-500, BNC811595-100, BNC811595-500, BNC001595-100, BNC001595-500, BNC701595-100, BNC701595-500, BNCH1595-100, BNCH1595-500, BNCP1595-250, BNUM1595-50, BNUB1595-100, BNUB1595-500. Aliquot and store in –20 ºC. ------------------------------ 2 ml. , BNCAP0074-500, BNCA0074-250, BNCB0074-100, BNCB0074-500, BNC060074-100, BNC060074-500, BNC050074-100, BNC050074-500, BNC040074-100, BNC040074-500, BNC880074-100, BNC880074-500, BNC140074-100, BNC140074-500, BNC430074-100, BNC430074-500, BNC550074-100, BNC550074-500, BNC680074-100, BNC680074-500, BNC940074-100, BNC940074-500, BNC400074-100, BNC400074-500, BNC470074-100, BNC470074-500, BNC600074-100, BNC600074-500, BNC610074-100, BNC610074-500, BNC800074-100, BNC800074-500, BNC810074-100, BNC810074-500, BNC700074-100, BNC700074-500, BNC000074-100, BNC000074-500, BNCH0074-100, BNCH0074-500, BNCP0074-250, BNUM0074-50, BNUB0074-100, BNUB0074-500, Fluorescent stains for cell surface, nucleus, mitochondria, lysosomes, & more. 8076.2) in 1 × MTSB; Calcofluor white (BR 28, Sigma, cat. The RedDot™ dyes are used to dye the nuclei of live cells, and they come in RedDot™ 1 and RedDot™ 2 options. Add the dye to complete culture medium. The following protocol is used for staining of //-->,

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