4 6 diamidino 2 phenylindole (dapi)

Manufactured by Fujifilm

Sourced in Japan, United States

DAPI is a fluorescent dye used for labeling and visualizing DNA in biological samples. It binds to the minor groove of double-stranded DNA, emitting a blue fluorescence when excited by ultraviolet light. DAPI is commonly used in microscopy and flow cytometry applications to stain cell nuclei.

Automatically generated - may contain errors

Lab products found in correlation

Lsm 700, by Zeiss (4 mentions) Pkh26, by Merck Group (3 mentions) Alexa488 conjugated goat anti mouse ig, by Thermo Fisher Scientific (3 mentions) Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (3 mentions) Paraformaldehyde (pfa), by Fujifilm (3 mentions) Alexa fluor 546, by Thermo Fisher Scientific (3 mentions) Axio observer z1, by Zeiss (3 mentions) Lsm 710 confocal microscope, by Zeiss (3 mentions) Bovine serum albumin (bsa), by Fujifilm (2 mentions) Biozero bz 8000, by Keyence (2 mentions) Triton x 100, by Fujifilm (2 mentions) Tcs sp5 2, by Leica camera (2 mentions) Bovine serum albumin (bsa), by Merck Group (2 mentions) Permafluor, by Thermo Fisher Scientific (2 mentions) Cy3 conjugated donkey anti rabbit igg secondary antibody, by Jackson ImmunoResearch (2 mentions) Zen 2009, by Zeiss (2 mentions) Paraformaldehyde phosphate buffer solution, by Fujifilm (2 mentions) Fv3000, by Olympus (2 mentions) Bz 2 analyzer, by Keyence (2 mentions) Bz x800, by Keyence (2 mentions)

78 protocols using 4 6 diamidino 2 phenylindole (dapi)

Immunohistochemical Analysis of Ext1 and Vimentin

Check if the same lab product or an alternative is used in the 5 most similar protocols

After cutting coronally at 5-μm thickness, the sections of human paraffin blocks were incubated with two primary antibodies—rabbit anti-Ext1 (dilution 1:50, Abcam, ab126305) and mouse anti-Vimentin (dilution 1:50, Dako, M0725)—overnight at 4°C. Then, the sections were incubated with Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594, dilution 1:300, Abcam, ab150084) and Goat Anti-Mouse IgG H&L (Alexa Fluor® 488, dilution 1:200, Abcam, ab150117), and finally stained with DAPI (dilution 1:1000, WAKO).
The sections of the mouse paraffin blocks were incubated with two primary antibodies, rabbit anti-Ext1 (dilution 1:100, Bioworld, BS6597), and chicken anti-Vimentin (dilution 1:100, Abcam, ab39376) overnight at 4°C. Then, the sections were incubated with Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594, dilution 1:300, Abcam, ab150084) and Goat Anti-Chicken IgY H&L (Alexa Fluor® 488, dilution 1:200, Abcam, ab150173), and finally stained with DAPI (dilution 1:1000, WAKO).

Niwa A., Taniguchi T., Tomita H., Okada H., Kinoshita T., Mizutani C., Matsuo M., Imaizumi Y., Kuroda T., Ichihashi K., Sugiyama T., Kanayama T., Yamaguchi Y., Sugie S., Matsuhashi N, & Hara A. (2023). Conditional ablation of heparan sulfate expression in stromal fibroblasts promotes tumor growth in vivo. PLOS ONE, 18(2), e0281820.

+ Open protocol

+ Expand

Immunofluorescence Analysis of Paraffin-Embedded Tissues

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Paraffin-embedded tissues were sectioned and used for the immunofluorescence analysis as previously described [25 (link),26 (link)]. Antigen retrieval was performed using low-pH EnVision FLEX Target Retrieval Solution (K8005, Dako, Omnis, Agilent, Santa Clara, CA, USA) according to the manufacturer’s protocol. The sections were blocked with PBS with 10% goat serum (G9023, Sigma-Aldrich, St. Louis, MO, USA) and 10% BSA (010-15153, Wako Pure Chemical Industries, Osaka, Japan) for 2 h at room temperature. After washing twice with PBS, primary antibodies were added (Table S1). The sections were then incubated at 4 °C overnight. These sections were washed six times with PBS and then incubated with the secondary antibody and 4’,6-diamidino-2-phenylindole (for staining of nuclei, 049-18801, Wako Pure Chemical Industries, Osaka, Japan) for 3 h in the dark at room temperature. The samples were then washed six times with PBS and mounted using a mounting medium (VECTASHIELD, H-1000, Vector Laboratories, Burlingame, CA, USA). The antibodies used and the reaction conditions are listed in Table S1. Immunohistochemical observations were performed using an Olympus DP73 fluorescence microscope with 40× objective lens. Images were captured using CellSens imaging software (Olympus, Tokyo, Japan).

Imamichi S., Chen L., Ito T., Tong Y., Onodera T., Sasaki Y., Nakamura S., Mauri P., Sanada Y., Igaki H., Murakami Y., Suzuki M., Itami J., Masunaga S, & Masutani M. (2022). Extracellular Release of HMGB1 as an Early Potential Biomarker for the Therapeutic Response in a Xenograft Model of Boron Neutron Capture Therapy. Biology, 11(3), 420.

+ Open protocol

+ Expand

Immunohistochemical Analysis of Muscle Tissue

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cross-sections of the midportion of the gastrocnemius were cut at 10 µm in a cryostat (Microm Cryo-Star HM 560, Walldorf, Germany) and maintained at −20 °C until analyses. The sections were fixed in 0.1 M phosphate buffer containing 4% paraformaldehyde for 5 min and degreased in 100% methanol for 10 min at −20 °C. After being washed by phosphate-buffer saline (PBS), the sections were blocked in 10% donkey serum diluted with PBS containing 0.1% Triton-X 100 (PBS-T) for 20 min. Then, the sections were incubated overnight at 4 °C with anti-Laminin α2 (1:600; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Pax7 (1:100; Developmental Studies Hybridoma Bank, Iowa City, IA, USA) and anti-MyoD (1:200; Santa Cruz Biotechnology) antibodies diluted in PBS-T containing 1% bovine serum albumin (BSA). Immunoreactivity was detected by incubation with Cy3-conjugated donkey anti-mouse IgG (1:500; Jackson ImmunoResearch, West Grove, PA, USA) and AlexaFluor 488-conjugated donkey anti-rabbit IgG (1:500; Life Technologies, Carlsbad, CA, USA) diluted in PBS-T containing 1% BSA for 4 h, Sections were counterstained with 4′,6-diamidino-2-phenylindole (Wako Pure Chemical Industries, Osaka, Japan). After several washes, the stained sections were mounted using the mounting medium (KPL, Gaithersburg, MD, USA). Images were acquired using an Olympus FV1000-D confocal microscope (Olympus, Tokyo, Japan).

Fujimaki S., Wakabayashi T., Asashima M., Takemasa T, & Kuwabara T. (2016). Treadmill running induces satellite cell activation in diabetic mice. Biochemistry and Biophysics Reports, 8, 6-13.

+ Open protocol

+ Expand

Hypoxia-induced HIF-1β Expression in Choriocarcinoma

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Three choriocarcinoma cell lines were cultured on FBS-coated coverslips. After incubation for 24 h under ambient or hypoxic conditions, these cells were fixed and permeabilized as previously reported (Sasagawa et al., 2018 (link)). For immunostaining of HIF-1β, the specimens were treated with the anti-HIF-1β monoclonal antibody (1:100, NB100-124; Novus Biologicals) for 1 h and then visualized using the Alexa Fluor 488-conjugated secondary antibody (1:500, A-11029; Invitrogen). The nuclei were counterstained with 4',6-diamidino-2-phenylindole (1:500, Wako Pure Chemicals, Osaka, Japan). The cell images were obtained using a fluorescence microscope (Biozero BZ-8000; Keyence, Osaka, Japan).

Sasagawa T., Nagamatsu T., Yanagisawa M., Fujii T, & Shibuya M. (2021). Hypoxia-inducible factor-1β is essential for upregulation of the hypoxia‐induced FLT1 gene in placental trophoblasts. Molecular Human Reproduction, 27(12), gaab065.

+ Open protocol

+ Expand

Immunostaining Cardiac Embryo Sections

Check if the same lab product or an alternative is used in the 5 most similar protocols

Embryos were embedded in optimum cutting temperature (OCT) compound (Sakura Tissue-Tek, Tokyo, Japan, 4583) after fixation and cut into 10–14 μm sections. After washing with PBS and blocking with 1% BSA in PBS for 1 h, sections were incubated with 500 × diluted primary antibodies, including anti-Smad4 (Santa Cruz, Dallas, TX, sc-7966), anti-Mypt1 (Proteintech, Rosemont, IL, 22117-1-AP), anti-Flag (DYKDDDDK)-Tag (Sigma Aldrich, St. Louis, MO, F3165 or Cell Signaling Technology, Danvers, MA, 8146S), anti-Myc Tag (Cell Signaling Technology 2278S), anti-Nkx2-5 (Abcam, Cambridge, UK, ab35842 or ab97355), and anti-TnT (Thermo, Waltham, MA, MS-295-P0) at 4 °C overnight. Sections were then incubated with 500 × diluted secondary antibodies conjugated with Alexa Fluor 488 or 555 (Molecular Probes, Eugene, OR). Nuclei were stained with 4, 6-diamidino-2-phenylindole (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan).

Hu W., Dong A., Karasaki K., Sogabe S., Okamoto D., Saigo M., Ishida M., Yoshizumi M, & Kokubo H. (2021). Smad4 regulates the nuclear translocation of Nkx2-5 in cardiac differentiation. Scientific Reports, 11, 3588.

+ Open protocol

+ Expand

Immunofluorescence Staining for ATBF1, Tubulin, and HA

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were fixed in 4 % paraformaldehyde in phosphate-buffered saline (PBS) at room temperature for 20 min, then washed with 0.25 % Triton-X in PBS, and blocked with 10 % normal goat serum. Cells were then incubated for 1 h at room temperature with primary antibodies against ATBF1 (D1-120; MBL), β-tubulin III (Sigma), and HA-tag (MBL). After three washes with 0.25 % Triton-X in PBS, cells were visualized using the following secondary antibodies: Alexa 488-conjugated goat anti-mouse, Alexa 546-conjugated goat anti-rabbit IgG, Alexa 488-conjugated goat anti-rat, and Alexa 546-conjugated goat anti-rat IgG (Molecular Probes, Eugene, OR, USA). Sections were incubated for 1 h in the dark, and after two washes with PBS, subjected to nuclear staining with 4′,6-diamidino-2-phenylindole (Wako, Osaka, Japan) for 5 min.

Kawaguchi M., Hara N., Bilim V., Koike H., Suzuki M., Kim T.S., Gao N., Dong Y., Zhang S., Fujinawa Y., Yamamoto O., Ito H., Tomita Y., Naruse Y., Sakamaki A., Ishii Y., Tsuneyama K., Inoue M., Itoh J., Yasuda M., Sakata N., Jung C.G., Kanazawa S., Akatsu H., Minato H., Nojima T., Asai K, & Miura Y. (2016). A diagnostic marker for superficial urothelial bladder carcinoma: lack of nuclear ATBF1 (ZFHX3) by immunohistochemistry suggests malignant progression. BMC Cancer, 16, 805.

+ Open protocol

+ Expand

Immunoblotting and Immunocytochemistry of Mouse eCS

Check if the same lab product or an alternative is used in the 5 most similar protocols

For immunoblotting and immunocytochemistry experiments, a rabbit polyclonal antibody (polyAb) against a synthetic peptide corresponding to the N-terminus (IYRNLYREDRNIEA) of the mouse eCS (GenBank accession no. NP_082221.2, 1/250 dilution for immunoblotting, 1/100 dilution for immunostaining) was produced by Japan Lamb Ltd (Hiroshima, Japan). Rabbit anti-CS polyAb (NE040/7S, 1/500 dilution for immunoblotting, 1/100 dilution for immunostaining) and anti-PLCz1 polyAb (ab181816, 1/250 dilution for immunoblotting, 1/50 dilution for immunostaining) were purchased from Nordic MUbio (Susteren, Netherlands) and Abcam Inc. (Cambridge, MA), respectively. Mouse anti-β-actin mAb (AC-15, 1/500 dilution for immunoblotting) and anti-glyceraldehyde-3-phosphate dehydrogenase mAb (5A12, 1/500 dilution for immunoblotting) were purchased from Sigma-Aldrich (St. Louis, MO) and WAKO Pure Chemical Industries (Osaka, Japan). IgGs conjugated with Alexa Fluor 488 and 546 were purchased from Molecular Probes (Invitrogen, Carlsbad, CA) as secondary antibodies for immunohistochemistry. Horseradish peroxidase-conjugated secondary Abs (Sigma-Aldrich) were used for immunoblotting. The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (WAKO Pure Chemical Industries, Osaka, Japan).

Kang W., Harada Y., Yamatoya K., Kawano N., Kanai S., Miyamoto Y., Nakamura A., Miyado M., Hayashi Y., Kuroki Y., Saito H., Iwao Y., Umezawa A, & Miyado K. (2019). Extra-mitochondrial citrate synthase initiates calcium oscillation and suppresses age-dependent sperm dysfunction. Laboratory Investigation; a Journal of Technical Methods and Pathology, 100(4), 583-595.

+ Open protocol

+ Expand

Immunofluorescence Microscopy of ACT1 in HeLa Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

HeLa cells seeded on a glass plate were fixed with 4% paraformaldehyde, permeabilized with PBS containing 1% Triton X-100 and reacted with rabbit anti-ACT1 antibody (1:100, H-300, Santa Cruz Biotechnology) overnight at 4°C. The cells were then reacted with an Alexa Fluor 488-conjugated F(ab′)2 fragment of goat anti-rabbit IgG (Invitrogen). Nuclei were stained with 4′,6-diamidino-2-phenylindole (Wako, Osaka, Japan). Images were obtained using a confocal laser-scanning microscope (FluoView FV10i; Olympus, Osaka, Japan). Images were collected with a ×60 water objective lens (numerical aperture = 1.3) (Olympus) and acquired using FV10-ASW software (Olympus).

Ohgakiuchi Y., Saino Y., Muromoto R., Komori Y., Sato A., Hirashima K., Kitai Y., Kashiwakura J.I., Oritani K, & Matsuda T. (2020). Dimethyl fumarate dampens IL-17-ACT1-TBK1 axis-mediated phosphorylation of Regnase-1 and suppresses IL-17-induced IκB-ζ expression. Biochemical and biophysical research communications, 521(4).

+ Open protocol

+ Expand

Immunofluorescence Imaging of Transfected COS-7 Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

COS-7 cells were seeded on collagen-coated glass bottom dishes (Matsunami Glass, Osaka Japan) and transfected with 500 ng of respective vector constructs. Twenty-four hours after transfection, the cells were treated with 10 nM E₂ (Merck, Kenilworth, NJ, USA) or 0.1% EtOH as a vehicle in DMEM with 2.5% charcoal-stripped FBS (Equitech-Bio) and 1% penicillin/streptomycin solution for another 24 h. Transfected and mock-transfected cells were washed three times with PBS and fixed in 10% formalin/PBS for 1 h. The fixed cells were permeabilized in 0.1% Triton X-100/PBS for 5 min and treated with 2H8 or PPZ0506 antibodies in 0.3% bovine serum albumin (BSA)/0.1% Tween-20/PBS for 16 h. After washing three times in 0.1% Tween-20/PBS, the cells were incubated with an Alexa Fluor 488-labeled goat anti-mouse IgG secondary antibody (Lot # 1975516; 1:250; Thermo Fisher Scientific) in 0.3% BSA/0.1% Tween-20/PBS for 1 h. Nuclei of cells were counterstained with DAPI (Fujifilm Wako). Immunofluorescent images were captured using a BZ-8000 Fluorescence Microscope System (Keyence, Osaka, Japan). Alexa Fluor 488 and DAPI images were pseudocolored in green and red, respectively.

Ishii H., Otsuka M., Kanaya M., Higo S., Hattori Y, & Ozawa H. (2019). Applicability of Anti-Human Estrogen Receptor β Antibody PPZ0506 for the Immunodetection of Rodent Estrogen Receptor β Proteins. International Journal of Molecular Sciences, 20(24), 6312.

+ Open protocol

+ Expand

Immunohistochemical Analysis of Alpha-Synuclein and Related Markers

Check if the same lab product or an alternative is used in the 5 most similar protocols

The sections were blocked by incubation with 4.5% normal goat serum (NGS) in PBS for one hour, then incubated overnight at 4 °C in PBS supplemented with 3% NGS, 0.2% Triton X-100 and one of the following antibodies: anti-alpha-synuclein (1:500; mouse; (211) Santa-Cruz), anti-phospho S129-alpha-synuclein (1:500; rabbit; EP1536Y, Abcam), anti-TH (1:1000; rabbit; Millipore), anti-HSC70 (1:500; mouse; B-6, Santa-Cruz), anti-LAMP2A (1:500; rabbit; EPR4207(2), Abcam), anti-GFAP (1:500; rabbit; Millipore), anti-S100β (1:100; rabbit; EP1576Y, Abcam), anti Iba1 (1:1000; rabbit; EPR16588), Abcam), anti-acetylated lysines (1:100; rabbit; Millipore). Sections were rinsed three times in PBS and incubated with Alexa-Fluor 488-labeled anti-mouse IgG or Alexa Fluor 594-labeled anti-rabbit IgG (Life Technology) for 1 h at room temperature. The sections were rinsed in PBS and the nuclei were counterstained with DAPI (dilution 1:10,000, Wako) for 3 minutes. The sections were mounted on glass slides and coverslips in Aqua-Poly/Mount (Polysciences), and stored at 4 °C in the dark until further processing. Images of the immunostained sections were taken within 48 h.

Francelle L., Outeiro T.F, & Rappold G.A. (2020). Inhibition of HDAC6 activity protects dopaminergic neurons from alpha-synuclein toxicity. Scientific Reports, 10, 6064.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!