Fixation permeabilization buffer

Manufactured by BioLegend

Fixation/permeabilization buffer is a solution used to prepare samples for intracellular staining and flow cytometry analysis. It serves to fix cells and permeabilize the cell membrane, allowing antibodies to access and bind to intracellular targets.

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Lab products found in correlation

Brefeldin a, by BioLegend (4 mentions) Cytoflex flow cytometer, by Beckman Coulter (4 mentions) Monensin, by BioLegend (4 mentions) Las x software, by Leica (2 mentions) Truestain fcx block, by BioLegend (2 mentions) Lsrfortessa cell analyzer, by BD (2 mentions) Facsvantage, by BD (1 mentions) Transcription factor staining kit, by Thermo Fisher Scientific (1 mentions) Rorγt, by BD (1 mentions) True nuclear transcription factor buffer set, by BioLegend (1 mentions) Novocyte flow cytometer, by Agilent Technologies (1 mentions) Rorγt, by BioLegend (1 mentions) Brefeldin a, by Merck Group (1 mentions) Ionomycin, by Merck Group (1 mentions) Anti foxp3, by BioLegend (1 mentions) Facsaria sorp flow cytometer, by BD (1 mentions) Anti cd8a percp cy5, by BioLegend (1 mentions) Anti il 17a pe, by Thermo Fisher Scientific (1 mentions) Anti il 10 apc, by BioLegend (1 mentions) Anti ifn γ bv 421, by BioLegend (1 mentions)

Close spelling variants of this product

Fixation and permeabilization buffer Fixation and Intracellular Staining Permeabilization Wash Buffer Nuclear Factor Fixation and Permeabilization Buffer Set Fixation/permeabilization wash buffer FOXP3 Fixation/Permeabilization Buffer Set Foxp3 fixation/permeabilization buffer kit Transcription factor fixation/permeabilization buffer FOXP3 Fixation/Permeabilization Buffer Permeabilization buffer Fixation buffer

14 protocols using fixation permeabilization buffer

Phenotypic Analysis of Regulatory T Cells

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Isolated peripheral blood mononuclear cells (PBMCs) were used for analysis of Treg phenotypes. Cells were surface stained with fluorescently coupled antibodies specific to human antigens CD3, CD4, CD25, CD127, CD45RO, CCR7 and CD39 (purchased from BioLegend) in staining buffer followed by fixation and permeabilization (Fixation/Permeabilization Buffer, BioLegend); and intracellularly stained with fluorescently coupled anti‐Foxp3 (BioLegend) antibody.
Mouse spleen cells were isolated for CD39^+/− Tregs phenotypes. Cells were surface stained with fluorescently coupled antibodies specific to mouse antigens CD4, CD25, CD39, CD44, CCR7, CD62L and LAG‐3 (purchased from BioLegend) in staining buffer followed by fixation and permeabilization (Fixation/Permeabilization Buffer, BioLegend); and intracellularly stained with fluorescently coupled anti‐Foxp3 and anti‐CTLA‐4 (BioLegend) antibody.
Flow cytometric data were acquired by using a FACSAria SORP flow cytometer (BD) and analysed by using FlowJo software, version 10.5.3 (Treestar).

Jin X., Lin T., Yang G., Cai H., Tang B., Liao X., Li H., Chen X., Gong L., Xu H., Sun Y., Tan P., Yin J., Ma H., Ai J., Wang K., Wei Q., Yang L, & Li H. (2020). Use of Tregs as a cell‐based therapy via CD39 for benign prostate hyperplasia with inflammation. Journal of Cellular and Molecular Medicine, 24(9), 5082-5096.

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Multiparametric Flow Cytometry Analysis

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Single cells were obtained from spleen and lymph-node of indicated mice. For cell surface analysis, 1∼5 × 10⁶ cells per sample were stained with Abs in the dark at 4°C for 30 min. After washing with cold FACS buffer (1 × PBS supplemented with 2% FBS), cells were analyzed using CytoFLEX flow cytometer (BECKMAN COULTER). Flowjo software (CytExpert) was used for data recorded and analyzed.
To analyze intracellular transcriptional factors, after a 30-min surface staining, cells were fixed and permeabilized according to the manual of Foxp3 kit, followed by anti-foxp3 antibody staining and FACS analysis.
For cytokine analysis, cell samples were stimulated in vitro with PMA/Ionomycin in the presence of Brefeldin A (BioLegend) and Monensin (BioLegend) for 4 h. Cells were washed and stained with anti-CD4 Abs. After a 30-min incubation and wash, cells were fixed and permeabilized using Fixation/Permeabilization buffer (BioLegend), and stained with IFN-γ, IL-4, and IL-9 antibodies and FACS analysis.

Lei L., Zhang X., Yang X., Su Y., Liu H., Yang H., Wang J., Zou Y., Wang X., Jiao A., Zhang C., Zheng H., Zhang J., Zhang D., Shi L., Zhou X., Sun C, & Zhang B. (2021). A Genetic Model Reveals Biological Features of Neonatal CD4 Helper Cells Undergone Homeostasis in Mice. Frontiers in Cell and Developmental Biology, 9, 659744.

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Intracellular IFN-β Quantification in Macrophages

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For intracellular cytokine staining, macrophages were stimulated in vitro with VSV for 8 hours, and protein transport inhibitor brefeldin A was added during the last 4 hours. Cells were collected and fixed with Fixation & Permeabilization Buffer (BioLegend). Then, cells were stained with intracellular IFN-β with anti-mouse IFN-β mAb-biotin (BioLegend), followed by secondary streptavidin-PE staining. Flow cytometry analyses were performed using FACSVantage (Becton Dickinson). Data were analyzed by FACSDiva.

Xia S., Tao Y., Cui L., Yu Y, & Xu S. (2019). MHC Class I Molecules Exacerbate Viral Infection by Disrupting Type I Interferon Signaling. Journal of Immunology Research, 2019, 5370706.

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Immunostaining and Confocal Imaging of Aorta

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Aorta were isolated and fixed in 4% paraformaldehyde/30% sucrose overnight and prepared by manually removing the perivascular adipose tissue, then lumen was exposed by cutting longitudinally along the greater curvature of the aortic arch. Samples were blocked and permeabilized in a 5% donkey serum, 1% Triton x-100, PBS buffer for 1 hour rotating at room temperature. Antibodies were diluted 1:500 and incubated overnight in 4-degrees Celsius. Samples were washed 3 times in 1 mL PBS, then stained with secondary antibody (1:1000 dilution) and further incubated at 4-degrees Celsius for 3 hours. Samples were washed 3 times in PBS and imaged by confocal microscopy. For Ki67-staining, aorta samples from C57BL/6 mice were freshly isolated, then incubated directly into fixation/permeabilization buffer (Biolegend, Cat 421403) for 1 hour rotating at room temperature. Samples were then washed in permeabilization buffer for an additional hour rotating at room temperature. Antibody staining was then performed, as described above. All images were collected using a 4-laser upright Leica SPE, 4-laser inverted Leica SP8, or a 7-laser inverted Leica SP8 microscopes, all with full spectral detectors. Image collection was performed using Leica LAS X software, and analysis was performed using Leica LAS X or Imaris (Bitplane) v8 and v9 software.

Williams J.W., Zaitsev K., Kim K.W., Ivanov S., Saunders B.T., Schrank P.R., Kim K., Elvington A., Kim S.H., Tucker C.G., Wohltmann M., Fife B.T., Epelman S., Artyomov M.N., Lavine K.J., Zinselmeyer B.H., Choi J.H, & Randolph G.J. (2020). Limited proliferation capacity of aorta intima resident macrophages requires monocyte recruitment for atherosclerotic plaque progression. Nature immunology, 21(10), 1194-1204.

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Comprehensive Single-Cell Analysis of Immune Cells

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Single immune cells were obtained from draining lymph nodes and tumor tissues of indicated mice. For cell surface analysis, a total of 1–5×10⁶ cells were stained with Abs in the dark at 4°C for 30 min. After washing with cold resuspension solution buffer (1×PBS supplemented with 2% FBS), cells were analyzed using CytoFLEX flow cytometer (Beckman Coulter). CytExpert software (V.2.4) was used for data analysis. To detect the expression of intracellular transcriptional factors, cells were fixed and permeabilized following 30 min surface staining according to the manual of Transcription Factor Staining kit (eBioscience), followed by anti-Foxp3 antibody staining and Flow Cytometry (FACS) analysis. For cytokine analysis, cell samples were stimulated in vitro with Phorbol 12-myristate 13-acetate/Ionomycin in the presence of Brefeldin A (BioLegend) and Monensin (BioLegend) for 4 hours. Cells were washed and stained with surface marker antibodies, fixed and permeabilized using Fixation/Permeabilization buffer (BioLegend), and stained with intracellular antibodies.

Zhang C., Lei L., Yang X., Ma K., Zheng H., Su Y., Jiao A., Wang X., Liu H., Zou Y., Shi L., Zhou X., Sun C., Hou Y., Xiao Z., Zhang L, & Zhang B. (2021). Single-cell sequencing reveals antitumor characteristics of intratumoral immune cells in old mice. Journal for Immunotherapy of Cancer, 9(10), e002809.

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Intracellular Staining and Flow Cytometry

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Staining was performed as previously described [7 (link),38 (link),41 (link)]. For intracellular staining of Foxp3, RORγt, and Helios, the isolated cells were stained with monoclonal antibodies (mAbs) against CD3e and CD4 (both from BioLegend, San Diego, CA, USA) and subsequently fixed with a True-Nuclear Transcription Factor Buffer Set (BioLegend). The cells were then stained with mAbs against Foxp3 (Thermo Fisher Scientific, Waltham, MA, USA), RORγt (BD Bioscience, Franklin Lakes, NJ, USA), and Helios (BioLegend), and then subjected to flow cytometry. For the intracellular cytokine staining, the LP cells were cultured for 5.5 h in a complete medium supplemented with 50 ng/mL PMA, 500 ng/mL ionomycin, and 5 μg/mL brefeldin A (Sigma, Burlington, MA, USA). The cells were harvested and stained with mAbs against cell surface antigens, followed by fixation in a fixation/permeabilization buffer (BioLegend). The fixed cells were subjected to intracellular staining, with mAbs, against IFNγ and TNFα (BioLegend). The stained samples were analyzed using a NovoCyte Flow Cytometer (ACEA Biosciences, San Diego, CA, USA) and the FlowJo software version 10 (TOMY Digital Biology, Tokyo, Japan).

Chudan S., Ishibashi R., Nishikawa M., Tabuchi Y., Nagai Y., Ikushiro S, & Furusawa Y. (2023). Effect of Wheat-Derived Arabinoxylan on the Gut Microbiota Composition and Colonic Regulatory T Cells. Molecules, 28(7), 3079.

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Lymphocyte Profiling of Lymph Nodes

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Single‐cell suspensions were prepared from ILN, BLN, ALN, CLN, and MLN, and stained with anti‐CD4, CD8, GP33, CD44, CD62L, and KLRG1 Abs in the dark at 4°C for 30 min. After washing with cold FACS buffer (1× PBS supplemented with 2% FBS), cells were analysed using CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA). CytExpert 2.4 was used for data analysis.
For cytokine analysis, lymphocytes from lymph nodes were stimulated in vitro with PMA/Ionomycin in the presence of Brefeldin A (BioLegend) and monensin (BioLegend) for 4 h in 37°C, 5% CO₂ environment. Cells were washed and stained with anti‐CD4, CD8, and GP33 antibodies. After a 30‐min incubation and FACS wash, cells were fixed and permeabilized using Fixation/ Permeabilization buffer (BioLegend), and stained with IFNγ, Perforin, and GranzymeB antibodies and FACS analysis.

Liu X., Sun Y., Su Y., Gao Y., Zhang T., Wang Q., Zhang X., Zhang D., Sun C., Li J., Li Z, & Zhang B. (2024). The compensatory role of T cells from lymph nodes in mice with splenectomy. Journal of Cellular and Molecular Medicine, 28(10), e18363.

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Isolation and Characterization of Skeletal Stem Cells

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Periosteal MSC were harvested as described from C57 Bl/6 and CD47-null mice. Post digestion, cells were resuspended in PBS and stained with a panel to mark skeletal stem cells^{24 (link),48 (link)} using conjugated antibodies for CD90, Sca1, CD140a, CD51, CD200, Ly51, CD105, and a lineage negative cocktail. Samples were stained with fixable viability dye for 30 minutes. TrueStain FcX block (Biolegend, San Diego, CA; 156604) was added and cells were pelleted and washed prior to antibody staining at 4 °C for 1 hour. Following staining, samples were pelleted, washed, and fixed using commercially available Fixation/Permeabilization Buffer (Biolegend, 426803). Cells were analyzed on an LSRFortessa Cell Analyzer (BD), and data was analyzed using FlowJo (BD).

Zondervan R.L., Capobianco C.A., Jenkins D.C., Reicha J.D., Fredrick L.M., Lam C., Isenberg J.S., Ahn J., Marcucio R.S, & Hankenson K.D. (2024). CD47 is Required for Mesenchymal Progenitor Proliferation and Fracture Repair. bioRxiv.

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Isolation and Characterization of Skeletal Stem Cells

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Periosteal MSC were harvested as described from C57 BI/6 and CD47-null mice. Post digestion, cells were resuspended in PBS and stained with a panel to mark skeletal stem cells^{24 (link),48 (link)} using conjugated antibodies for CD90, Sca1, CD140a, CD51, CD200, Ly51, CD105, and a lineage negative cocktail. Samples were stained with fixable viability dye for 30 minutes. TrueStain FcX block (Biolegend, San Diego, CA; 156604) was added and cells were pelleted and washed prior to antibody staining at 4 °C for 1 hour. Following staining, samples were pelleted, washed, and fixed using commercially available Fixation/Permeabilization Buffer (Biolegend, 426803). Cells were analyzed on an LSRFortessa Cell Analyzer (BD), and data was analyzed using FlowJo (BD).

Hankenson K., Zondervan R., Capobianco C., Jenkins D., Reicha J., Frederick L., Lam C., Isenberg J., Ahn J, & Marcucio R.S. (2024). CD47 is Required for Mesenchymal Progenitor Proliferation and Fracture Repair. Research Square.

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Murine Th Cell Cytokine Profiling

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Femoral bone marrow cells were isolated and plated, consistent with methods reported for In Vitro Assays. Whole marrow cultures were maintained undisturbed for 4-hours; cultures were subsequently stimulated for 1-hour with 1 µl/ml PMA and 2 µl/ml Ionomycin, followed by 4-hours stimulation with 1 µl/ml Monensin. Cells were harvested, washed, and counted. Surface stains were performed and intracellular stains were carried out following the fixation-permeabilization buffer manufacturer’s protocol (BioLegend): anti-CD3e-FITC (Tonbo Biosciences, clone 145-2C11), anti-CD4-APC/Cy7 (BD Pharmingen, clone GK1.5), anti-CD8a-PerCP/Cy5.5 (BioLegend, clone 53-6.7), anti-IL10-APC (BioLegend, clone JES5-16E3), anti-IL17a-PE (eBioscience, clone eBio17B7), anti-IFNγ-BV421 (BioLegend, clone XMG1.2). Data was acquired by the FACSVerse System (BD Biosciences). Analyses were performed via FlowJo VX software.

Novince C.M., Whittow C.R., Aartun J.D., Hathaway J.D., Poulides N., Chavez M.B., Steinkamp H.M., Kirkwood K.A., Huang E., Westwater C, & Kirkwood K.L. (2017). Commensal Gut Microbiota Immunomodulatory Actions in Bone Marrow and Liver have Catabolic Effects on Skeletal Homeostasis in Health. Scientific Reports, 7, 5747.

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