Blocking one

Manufactured by Nacalai Tesque

Sourced in Japan, United States, United Kingdom, Germany

Blocking One is a laboratory equipment product designed for general use in scientific research and experimentation. Its core function is to provide a physical barrier or obstruction to prevent the flow or movement of materials or substances within a controlled environment. The specific intended use or application of this product is not provided.

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Lab products found in correlation

Pvdf membrane, by Merck Group (20 mentions) Ecl prime western blotting detection reagent, by GE Healthcare (20 mentions) Chemi lumi one super, by Nacalai Tesque (18 mentions) Protease inhibitor cocktail, by Nacalai Tesque (17 mentions) Las 4000, by Cytiva (13 mentions) Polyvinylidene difluoride membrane, by Merck Group (13 mentions) Blocking one p, by Nacalai Tesque (13 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (12 mentions) Can get signal, by Toyobo (12 mentions) Polyvinylidene difluoride membrane, by Bio-Rad (12 mentions) Las4000, by GE Healthcare (11 mentions) Bz 9000, by Keyence (10 mentions) Hybond, by Cytiva (10 mentions) Bca protein assay kit, by Thermo Fisher Scientific (10 mentions) Immobilon p, by Merck Group (9 mentions) Las 4000, by Fujifilm (9 mentions) Mini protean tgx gel, by Bio-Rad (8 mentions) Pvdf membrane, by Bio-Rad (8 mentions) Image lab software, by Bio-Rad (8 mentions) Imagequant las 4000 mini, by GE Healthcare (8 mentions)

Close spelling variants of this product

Blocking One Histo (122 citations) Blocking One solution (79 citations) Blocking One-P (69 citations) Blocking One reagent (56 citations) Bullet Blocking One (25 citations) Blocking One buffer (7 citations) Bullet Blocking One for Western Blotting (7 citations) Blocking One-P solution (7 citations) Bullet Blocking One reagent (5 citations) Blocking One-P reagent (4 citations)

457 protocols using blocking one

Immunofluorescence Analysis of Hepatic Lineage Markers

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Cells were fixed in 4% paraformaldehyde (Nacalai Tesque) in PBS, permeabilized with 0.1% Triton X-100 (Nacalai Tesque), and were then blocked with 20% Blocking One (Nacalai Tesque, Japan) in PBST (0.1% Tween-20 in PBS). Antibodies were diluted in 20% Blocking One (Nacalai Tesque, Japan) in PBST (0.1% Tween-20 in PBS). Cells were counterstained with 6-diamidino-2-phenylindole (DAPI) (Roche Diagnostics, Switzerland).
The following antibodies were used: rabbit anti-AFP (Dako, Glostrup, Denmark), goat anti-ALB (Bethyl), mouse anti-OCT3/4 (Santa Cruz), goat anti-SOX17 (R&D Systems), and Alexa 568-conjugated and Alexa 488-conjugated antibodies (Invitrogen). Positive cells versus total cells (DAPI-positive cells) were quantified using the Metamorph Image Analysis Software (Molecular Devices).

Nakai S., Shibata I., Shitamichi T., Yamaguchi H., Takagi N., Inoue T., Nakagawa T., Kiyokawa J., Wakabayashi S., Miyoshi T., Higashi E., Ishida S., Shiraki N, & Kume S. (2019). Collagen vitrigel promotes hepatocytic differentiation of induced pluripotent stem cells into functional hepatocyte-like cells. Biology Open, 8(7), bio042192.

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Myosin Heavy Chain Immunofluorescence Assay

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Cells were cultured on µ-Plate 24 Well (ibidi), washed twice with phosphate-buffered saline (PBS), fixed with 1% paraformaldehyde in PBS, permeabilized with 0.5% Triton X-100 in PBS and washed twice with PBS. A 15 min incubation with Blocking One (Nacalai Tesque Inc.) was followed by a 2 h incubation with mouse anti-myosin heavy chain (MF20, eBioscience, 1 : 200) diluted with 10% Blocking One in PBS at room temperature. The µ-Plate 24 Well was then washed three times with PBS and incubated for 30 min at room temperature with CF568-labelled goat anti-mouse antibody (1 : 1000; Biotium Inc.) and Bisbenzimide H33342 Fluorochrome Trihydrochloride (Hoechst) (1 : 2000; Nacalai Tesque Inc.) diluted with 10% Blocking One in PBS. The µ-Plate 24 Well was again washed three times in PBS and mounted in ibidi Mounting Medium (ibidi). Images were visualized using a fluorescence microscope (BZ-9000; Keyence). Co-localization was evaluated using BZ-II Analyzer software (Keyence).

Kudou K., Komatsu T., Nogami J., Maehara K., Harada A., Saeki H., Oki E., Maehara Y, & Ohkawa Y. (2017). The requirement of Mettl3-promoted MyoD mRNA maintenance in proliferative myoblasts for skeletal muscle differentiation. Open Biology, 7(9), 170119.

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Immunofluorescence Staining of Paraffin-Embedded Tissue

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Paraffin-embedded sections (10 µm) were mounted on glass slides. The sections were deparaffinized with xylene, dehydrated
with ethanol and subsequently incubated with HistoVT One (Nacalai Tesque, Kyoto, Japan) at 90 C for 30 min to mediate antigen
retrieval. The sections were then washed with distilled water, incubated with Blocking One (Nacalai Tesque) at 4 C for 1 h
and subsequently incubated with primary antibodies at 4 C overnight [the primary antibodies were added to Blocking One
(Nacalai Tesque) and phosphate-buffered saline-mixed liquor (diluted 1:200)]. After incubation, the sections were incubated
with Alexa Fluor 488-labeled anti-mouse and Alexa Fluor 594-labeled anti-rabbit secondary antibodies (diluted 1:1000) at 4 C
for 3 h. The nuclei were counterstained with Hoechst 33342 (diluted 1:5000; Molecular Probes, Eugene, OR, USA). The stained
images were obtained using an LSM-700 confocal laser microscope (Carl Zeiss; Oberkochen, Germany), and the fluorescent
brightness was analyzed with the ZEN2010 software in conjunction with the LSM-700 microscope. The stage of each seminiferous
tubule was determined following the criteria described previously [24 ].

SHIRAKATA Y., HIRADATE Y., INOUE H., SATO E, & TANEMURA K. (2014). Histone H4 Modification During Mouse Spermatogenesis. The Journal of Reproduction and Development, 60(5), 383-387.

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Immunohistochemical Analysis of Intestinal Immune Cells

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Jejunum, ileum including distal PPs and colon samples were collected as described above, and were fixed immediately in 1.0% paraformaldehyde and 0.1M L-Lysine (Sigma-Aldrich, St. Louis, MO) add to PBS (pH7.4), incubated in 20% sucrose at 4°C, embedded in tissue-tek OCT compound (Sakura Finetek USA, Inc., Torrance, CA), and stored at −80°C until further use. Thin 5-8μm-thick sections were cut with Leica CM 1950 cryostat (Leica Microsystems GmbH, Wetzlar, Germany), permeabilized in PBS containing 0.1% Triton X-100 (Sigma-Aldrich) and 20% Blocking One (Nacalai tesque, Kyoto, Japan), incubated in with fluorochrome-conjugated antibodies (anti-human CD4 (OKT4), CD8 (HIT8a), CD45RA (HI100), Foxp3 (259D), and DAPI) in blocking buffer (PBS + 20% blocking reagent; Blocking One) at room temperature. Samples were mounted on cover slips with Prolong Diamond Antifade Mountant (Life Technologies, Inc., Gaithersburg, MD). Imaging was performed on an EVOS FL Auto 2 Imaging System (Invitrogen) with motorized z focus stage for fully automated image stitching. Specific cells were identified using the “spots” command in Imaris software (Bitplane, Zurich, Switzerland), and nonspecific fluorescence signals were manually removed. The lamina propria lesion and ILFs area were defined using ImageJ software (National Institutes of Health, Bethesda, MD).

Senda T., Dogra P., Granot T., Furuhashi K., Snyder M.E., Carpenter D.J., Szabo P.A., Thapa P., Miron M, & Farber D.L. (2018). Microanatomical dissection of human intestinal T-cell immunity reveals site-specific changes in gut-associated lymphoid tissues over life. Mucosal immunology, 12(2), 378-389.

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Immunohistochemical Analysis of Intestinal Immune Cells

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Western Blot Protocol for Protein Analysis

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Samples were separated by SDS-PAGE using a 7.5% acrylamide gel and transferred to nitrocellulose membranes (GE Healthcare) for 1 h at 100 V and 4 °C. The membranes were treated with Blocking One (Nacalai Tesque) for 30 min at room temperature and then incubated sequentially with primary and secondary antibodies diluted in Blocking One in PBST. Detection was performed with SIGMAFAST BCIP/NBT (Sigma-Aldrich).
Uncropped blots are shown in (Fig. S7).

Yamashita R., Fujii S., Ushioda R, & Nagata K. (2021). Ca2+ imbalance caused by ERdj5 deletion affects mitochondrial fragmentation. Scientific Reports, 11, 20772.

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Immunofluorescence Staining of Mitochondria and LC3

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Cells were fixed with PBS containing with 2% (w/v) paraformaldehyde for 10 min at 4 °C. Fixed samples were blocked with Blocking One (Nacalai Tesque) for 45 min at 4 °C and incubated overnight at 4 °C with primary antibodies diluted in 10% (v/v) Blocking One in PBS-T (PBS with 0.2% (v/v) Triton X-100 solution (Nacalai Tesque)). The samples were then washed 3 times with PBS-T and incubated for 1 h at room temperature with secondary antibodies diluted in 10% (v/v) Blocking One in PBS-T. Mitochondria were stained with MitoTracker Red CMXRos (Thermo Fisher Scientific) according to the manufacturer’s instructions. Nuclei were stained with DAPI (1:5000; Sigma-Aldrich) or TO-PRO-3 (1:1000; Thermo Fisher Scientific). The samples were observed with BZ-X700 (KEYENCE, Osaka, Japan) or LSM710NLO confocal microscope (Carl Zeiss, Oberkochen, Germany). The MFI of MitoTracker Red or LC3 in MHC-positive area was calculated using BZ-X Analyzer software (KEYENCE). Antibodies are listed in Supplementary Table S2.

Yoshida T., Awaya T., Jonouchi T., Kimura R., Kimura S., Era T., Heike T, & Sakurai H. (2017). A Skeletal Muscle Model of Infantile-onset Pompe Disease with Patient-specific iPS Cells. Scientific Reports, 7, 13473.

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Western Blotting with Inhibitors

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Cells were lysed in sodium dodecyl sulfate (SDS)–sample buffer containing protease and phosphatase inhibitors (2 µg/mL aprotinin, 0.8 µg/mL pepstatin A, 2 µg/mL leupeptin, 2 mM PMSF, 20 mM β-glycerophosphate, 50 mM NaF, and 10 mM Na₃VO₄). The cell lysates were subjected to SDS–polyacrylamide gel electrophoresis and were electrotransferred onto polyvinylidene difluoride membranes (PVDF; Pall Corporation, Port Washington, NY, USA). After blocking with Blocking One (Nacalai Tesque) at room temperature for 30 min, the membranes were incubated with the antibodies diluted with Tris-buffered saline [20 mM Tris–HCl (pH 7.5), 137 mM NaCl] containing 0.1% Tween 20 and 5% Blocking One. Chemiluminescence was detected with ChemiDoc XRSplus image analyzer (Bio-Rad) using Clarity (Bio-Rad) as the chemiluminescence substrate.

Ikeda Y., Yasutake R., Yuki R., Saito Y, & Nakayama Y. (2021). Combination Treatment of OSI-906 with Aurora B Inhibitor Reduces Cell Viability via Cyclin B1 Degradation-Induced Mitotic Slippage. International Journal of Molecular Sciences, 22(11), 5706.

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Immunocytochemistry for MHC expression

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Cells were plated in 24-well plates (ibidi USA Inc., WI, USA), washed twice with phosphate-buffered saline (PBS), fixed with 1% paraformaldehyde in PBS for 10 min, permeabilized with 0.5% Triton X-100 in PBS, and washed twice with PBS. A 15 min incubation with Blocking One (Nacalai Tesque Inc.) was followed by a 2 h incubation with mouse anti-MHC (Calbiochem, 1:100; Fig. 1d) diluted with 10% Blocking One in PBS at room temperature. The wells were then washed three times with PBS and incubated for 30 min at room temperature with CF568-labeled goat anti-mouse (1:1000; Biotium Inc.) and bisbenzimide H33342 fluorochrome trihydrochloride (Hoechst) (1:5000; Nacalai Tesque Inc.) diluted with 10% Blocking One in PBS. Wells were washed three times in PBS and mounted in ibidi mounting medium (ibidi USA, Inc.). Images were visualized using a fluorescence microscope (BZ-9000; Keyence). The fusion index was evaluated using the ImageJ software.

Harada A., Maehara K., Ono Y., Taguchi H., Yoshioka K., Kitajima Y., Xie Y., Sato Y., Iwasaki T., Nogami J., Okada S., Komatsu T., Semba Y., Takemoto T., Kimura H., Kurumizaka H, & Ohkawa Y. (2018). Histone H3.3 sub-variant H3mm7 is required for normal skeletal muscle regeneration. Nature Communications, 9, 1400.

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Larval Protein Extraction and Western Blotting

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Larvae were homogenized with a Dounce tissue grinder and lysed with lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% NP-40, 0.1% SDS) containing protease inhibitor cocktail (Roche). Lysates were mixed with 5× Laemmli sampling buffer containing 100 mM DTT and boiled at 95°C for 3 min. Proteins were separated by SDS-PAGE and transferred onto PVDF membranes (Merck Millipore). After blocking with BLOCKING ONE (Nacalai Tesque), the membranes were incubated with primary antibodies in phosphate-buffered saline containing 0.1% Tween 20 and 10% BLOCKING ONE, followed by incubation with horseradish peroxidase-conjugated secondary antibodies. Signals were detected with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) and images were captured by ImageQuanta LAS 4000 (GE Healthcare Bio-Sciences).

Guo L., Yamashita H., Kou I., Takimoto A., Meguro-Horike M., Horike S.I., Sakuma T., Miura S., Adachi T., Yamamoto T., Ikegawa S., Hiraki Y, & Shukunami C. (2016). Functional Investigation of a Non-coding Variant Associated with Adolescent Idiopathic Scoliosis in Zebrafish: Elevated Expression of the Ladybird Homeobox Gene Causes Body Axis Deformation. PLoS Genetics, 12(1), e1005802.

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