Citrate buffer ph 6

Manufactured by Thermo Fisher Scientific

Sourced in Germany, United States

Citrate buffer pH 6 is a solution that maintains a pH of 6. It is a common buffer used in various laboratory applications, such as protein purification, enzyme assays, and cell culture experiments.

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Lab products found in correlation

Decloaking chamber, by Biocare Medical (3 mentions) Dab substrate, by Agilent Technologies (2 mentions) Harris modified hematoxylin, by Merck Group (2 mentions) Clone 1f6, by Abcam (2 mentions) Hrp conjugated anti mouse secondary antibody, by Agilent Technologies (2 mentions) Tissuefaxs whole slide scanning system, by TissueGnostics (2 mentions) Histoquest software, by TissueGnostics (2 mentions) Fluorescent microscope, by Leica (1 mentions) Peroxidase histostainplus kit, by Thermo Fisher Scientific (1 mentions) Vimentin, by BD (1 mentions) Tdt in situ apoptosis detection kit, by R&D Systems (1 mentions) Nitrotyrosine, by Merck Group (1 mentions) Dab substrate, by Vector Laboratories (1 mentions) Imagej, by PerkinElmer (1 mentions) Cell profiler, by PerkinElmer (1 mentions) Opera phenix high content screening system, by PerkinElmer (1 mentions) Yopro, by Thermo Fisher Scientific (1 mentions) Cy3 donkey anti rabbit serum, by Dianova (1 mentions) Zen 2.3 blue edition software, by Zeiss (1 mentions) Lsm 800 confocal laser scanning microscope, by Zeiss (1 mentions)

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Citrate buffer (60 citations)

14 protocols using citrate buffer ph 6

Multiplexed Immunohistochemistry for Immune Profiling

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For immunohistochemistry, paraffin-embedded sections were de-paraffinized in xylene and rehydrated in graded alcohol. Antigen retrieval was done by incubating the sections in citrate buffer pH 6 (Invitrogen) by either boiling for 10 min or in the microwave. Staining was performed using peroxidase HistostainPlus Kit (Invitrogen) according to the manufacturer's protocol. For fluorescent staining, cells were fixed with 4% paraformaldehyde at for 10 min. After rehydrating in PBS, cells were incubated with primary antibodies at room temperature for an hour, washed and incubated 30 min with fluorescence-conjugated secondary antibodies. The nuclei were stained with DAPI/antifade (Invitrogen) and coverslipped. Sections were examined with a fluorescent microscope (Leica). The following primary antibodies were used: Ly6C (#ab15627-Dilution 1/100, Abcam), Ly6G (#MAB1037-Dilution 1/100, R&D biosystem), Ki67 (#12202-Dilution 1/100, Cell Signaling), Vimentin (#550513 Dilution 1/100, BD Biosciences), CD14 (#10073-H08H Dilution 1/100, Sino biological). The secondary antibodies were purchased from Invitrogen.

Ouzounova M., Lee E., Piranlioglu R., El Andaloussi A., Kolhe R., Demirci M.F., Marasco D., Asm I., Chadli A., Hassan K.A., Thangaraju M., Zhou G., Arbab A.S., Cowell J.K, & Korkaya H. (2017). Monocytic and granulocytic myeloid derived suppressor cells differentially regulate spatiotemporal tumour plasticity during metastatic cascade. Nature Communications, 8, 14979.

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Immunohistochemistry and TUNEL Assay for Apoptosis

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For immunohistochemistry, paraffin-embedded sections were de-paraffinized in xylene and rehydrated in graded alcohol. Antigen retrieval was done by incubating the sections in citrate buffer pH 6 (Invitrogen) using microwave. Staining was performed using Peroxidase Detection Kit (#1859346-Thermo scientific) with pan-keratin (# PM162AA) antibody (Biocare Medical) according to the manufacturer’s protocol. For the TUNEL assay, TdT In Situ Apoptosis Detection Kit (#4811–30-K) were used according to the manufacturer’s recommendation (R&D System). Briefly, rehydrated lung tissue slides were incubated with Proteinase K solution for 15 min and then washed with deionized water before immersion in quenching solution for 5 min. Following the incubation with 1X TdT labeling buffer for 5 min, tissues were kept in labeling reaction mix for 60 min at 37 ^oC. The reaction was stopped and samples were washed twice with PBS for 5 min. Finally, the slides were incubated with horseradish peroxidase-conjugated streptavidin at 37 °C for 10 min and immersed in Blue label solution for 5 min. After in situ labeling, tissues were counterstained with nuclear fast red, dehydrated, and mounted. The average number of apoptotic cells were calculated by counting three independent area under bright-field microscope.

Piranlioglu R., Lee E., Ouzounova M., Bollag R.J., Vinyard A.H., Arbab A.S., Marasco D., Guzel M., Cowell J.K., Thangaraju M., Chadli A., Hassan K.A., Wicha M.S., Celis E, & Korkaya H. (2019). Primary tumor-induced immunity eradicates disseminated tumor cells in syngeneic mouse model. Nature Communications, 10, 1430.

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Oxidative Stress Markers in IL-13 Lung Tissue

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γH2A.X, 8-Hydroxyguanosine, and Nitrotyrosine stains were done on lung tissue of (WT) and IL-13 mice. The slides were placed in xylene to remove paraffin, then a series of ethanol washes. After a wash in tap water, the slides were incubated in 3% Hydrogen peroxide / methanol solution for 10 minutes. The slides were then washed in distilled water, and incubated for 25 minutes in Citrate Buffer pH6 (Invitrogen Corporation) at 95 degrees Celsius using a vegetable steamer. Next, the slides were brought to room temperature, rinsed with PBST (Phosphate Buffered Saline containing 0.05% Tween-20), then incubated at room temperature for 1 hour with Anti-gamma H2A.X (phosphor S139) antibody (Abcam, ab22551), 2 hours with anti-8-Hydroxyguanosine antibody (Abcam, ab48508), or for 45 minutes with Nitrotyrosine (Millipore, 06-284) at the dilutions of 1: 50 for γH2A.X, and 8-Hydroxyguanosine antibodies and at1:200 for the Nitrotyrosine antibody, respectively. The slides were then rinsed with PBST, and were incubated with Dako EnVision+ System –HRP Labelled Polymer Anti-Mouse (Dako, K4001) at room temperature for 30 minutes. Subsequently after a rinse with PBST, the slides were incubated with DAB (3,3′-Diaminobenzidine) for visualization. Finally, the slides were washed in tap water, counterstained with Harris' Hematoxylin, dehydrated in ethanol, and mounted with media.

Chapman A.M., Malkin D.J., Camacho J, & Schiestl R.H. (2014). IL-13 Overexpression in Mouse Lungs Triggers Systemic Genotoxicity in Peripheral Blood. Mutation research, 769, 100-107.

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Immunohistochemical Analysis of CD44 and CD146 in Breast Tissue

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Paraffin blocks including normal and tumor breast tissue were obtained from the archives of the Department of Pathology at Louisiana State University Health Sciences Center, New Orleans. Immunohistochemical assays were performed by examining adjacent sections for both CD44 and CD146 expression using mouse anti-human CD44 antibody (1:100 dilution, R&D, CA) and polyclonal mouse anti-CD146 antibody (1:25 dilution, Novocastra) after antigen retrieval carried out by boiling the samples in 500 ml of 9 mM citrate buffer (pH 6) (Invitrogen, Carlsbad, CA) for 25 min. Primary antibodies were applied overnight at 4°C. Biotinylated secondary antibody of vector labs VECTASTAIN ABC systems universal kit and DAB substrate were applied according to the manufacturers specifications (Vector labs, CA). For the intensity of immunostaining, we adopted a simple comparison of the intensity of immunostaining using 1+ for low expression, 2+ for intermediate expression and 3+ for high expression.

Ouhtit A., Abdraboh M.E., Hollenbach A.D., Zayed H, & Raj M.H. (2017). CD146, a novel target of CD44-signaling, suppresses breast tumor cell invasion. Cell Communication and Signaling : CCS, 15, 45.

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Quantification of HIV-1 Infection in Mice

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Formalin fixed, paraffin embedded tissue from lung and spleen samples of HIV-1 infected and uninfected mice were cut in 4 μm sections. Antigen retrieval was carried out in a Decloaking Chamber (BioCare Medical), in citrate buffer pH 6 (Invitrogen). Sections were incubated with anti-HIV-1 p24 (1:50; clone KC57 Beckman Coulter) or anti-human CD4 (1:50; clone 1F6 Abcam) antibody overnight at 4°C. Slides were washed five times in Tris buffered saline with 0.05% Tween-20 (TBST). Secondary anti-mouse HRP conjugated antibody (Dako) was applied for 1 hr at RT. After another five washes with TBST, staining was visualized using DAB substrate (Dako). Tissue samples were counterstained with Harris Modified Hematoxylin (Sigma). Whole tissue slide sections were scanned with an Olympus TissueFAXS whole slide scanning system and the number of p24+ or CD4+ cells were quantified using HistoQuest software (TissueGnostics) and by taking the average of 2 consecutive tissue sections from per sample. Background was subtracted for each sample by staining 2 consecutive tissue sections without the addition of the secondary antibody. The average number of unspecific stained cells from 2 slides were subtracted from the average number of CD4+ or p24+ cells.

Corleis B., Bucsan A.N., Deruaz M., Vrbanac V.D., Lisanti-Park A.C., Gates S.J., Linder A.H., Paer J.M., Olson G.S., Bowman B.A., Schiff A.E., Medoff B.D., Tager A.M., Luster A.D., Khader S.A., Kaushal D, & Kwon D.S. (2019). HIV-1 and SIV Infection Are Associated with Early Loss of Lung Interstitial CD4+ T Cells and Dissemination of Pulmonary Tuberculosis. Cell reports, 26(6), 1409-1418.e5.

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Quantification of HIV-1 Infection in Mice

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Immunofluorescence Staining Protocol

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Cultures were fixed with 2% PFA for 20 min at room temperature (RT), rinsed with PBS, and antigen retrieved in citrate buffer pH 6.0 (ThermoFisher) in water bath at 80 °C for 5 min. Samples were left to rest for 10 mins at room temperature before being permeabilised and blocked in PBS, 10% donkey serum and 0.01% of Triton X-100 for 10 min. Incubation of primary antibody in PBS and 10% donkey serum was performed overnight at 4 °C. Samples were washed with PBS and subsequently incubated in species-appropriate Alexa Fluor© secondary antibody in PBS with 10% donkey serum for 1 h at RT. The list of primary and secondary antibodies used for immunostaining is available on Zenodo (10.5281/zenodo.10650790). Images were acquired on the Opera Phenix High-content Screening system (PerkinElmer) or the Invitrogen EVOS ™ FL Auto (ThermoFisher) cell imaging system and subsequently processed on Harmony (Perkin Elmer; RRID:SCR_018809), ImageJ (RRID:SCR_003070) or CellProfiler (RRID:SCR_007358), respectively.

Do Q.B., Noor H., Marquez-Gomez R., Cramb K.M., Ng B., Abbey A., Ibarra-Aizpurua N., Caiazza M.C., Sharifi P., Lang C., Beccano-Kelly D., Baleriola J., Bengoa-Vergniory N, & Wade-Martins R. (2024). Early deficits in an in vitro striatal microcircuit model carrying the Parkinson’s GBA-N370S mutation. NPJ Parkinson's Disease, 10, 82.

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Immunofluorescence Analysis of CRC Samples

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CRC patients undergoing surgery as a primary treatment were included in the study. Previous neo-adjuvant treatment or chemoradiotherapy were the exclusion criteria. The study was approved by the Ethics Committee of the Medical Faculty of the University of Tuebingen (confirmation #426/2013BO1). All patients provided signed, informed consent. Paraffin sections were deparaffinized and rehydrated. Slides were incubated in citrate buffer pH 6.0 (Thermo Scientific, Karlsruhe, Germany) using a pressure cooker. For immunofluorescence analysis, sections were blocked with donkey serum followed by incubation with rabbit antiserum to phospho-YB-1 and mouse antiserum to Ki67. The bound antibody was visualized by incubation with Cy3-donkey anti-rabbit serum and Cy5-donkey anti-mouse serum (Dianova, Hamburg, Germany). Nuclei were stained with Yopro (1:2000 Invitrogen, Karlsruhe, Germany). Sections were analyzed using a Zeiss LSM 800 confocal laser scanning microscope, with Zeiss ZEN 2.3 (blue edition) Software.

Maier E., Attenberger F., Tiwari A., Lettau K., Rebholz S., Fehrenbacher B., Schaller M., Gani C, & Toulany M. (2019). Dual Targeting of Y-Box Binding Protein-1 and Akt Inhibits Proliferation and Enhances the Chemosensitivity of Colorectal Cancer Cells. Cancers, 11(4), 562.

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Prostate Carcinoma Cell Line Characterization

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Stable human maspin-transfected (M7) or mock-transfected control (Neo) cells, derived from the human prostate carcinoma cell line DU145 (American Type Culture Collection, Manassas, VA), were cultured as previously described [14 (link)]. The reagents and kits used in this study include: hematoxylin, eosin, and accustain Masson's trichrome (Sigma-Aldrich, St. Louis, MO); TMB (3,3′,5,5′-tetramethylbenzidine) substrate kit and DiffQuick staining kit (Fisher Scientific, Pittsburgh, PA); citrate buffer pH 6 (Life Technologies, Grand Island, NY), vectastain ABC kit (Vector Labs, Burlingame, CA), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay kit (Millipore, Billerica, MA) and mono-poly resolving medium (MP Biomedical, Santa Ana, CA).

Dzinic S.H., Chen K., Thakur A., Kaplun A., Daniel Bonfil R., Li X., Liu J., Margarida Bernardo M., Saliganan A., Back J.B., Yano H., Schalk D.L., Tomaszewski E.N., Beydoun A.S., Dyson G., Mujagic A., Krass D., Dean I., Mi Q.S., Heath E., Sakr W., Lum L.G, & Sheng S. (2014). Maspin expression in prostate tumor elicits host anti-tumor immunity. Oncotarget, 5(22), 11225-11236.

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Mouse Nudt7 cDNA Expression and Analysis

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The cDNA clone (ID 5102473) carrying the full-length sequence of mouse Nudt7 was purchased from GE Healthcare; pmCHERRY-C1 vector from Clontech; the HEK293 cell line was from the American Type Culture Collection; pET-28a(+) from EMD Millipore; oligonucleotides, pCR2.1 vector, RNAlater, Pluronic F-68, Lipofectamine 2000, cell culture reagents and citrate buffer pH 6 were from Life Technologies; restriction enzymes were from New England BioLabs. Lipid standards were purchased from Sigma-Aldrich or Avanti Polar Lipids and high performance thin layer chromatography (HPTLC) silica gel 60 plates from EMD Millipore. All other reagents were purchased from Sigma-Aldrich or Fisher Scientific, unless otherwise stated.

Shumar S.A., Fagone P., Alfonso-Pecchio A., Gray J.T., Rehg J.E., Jackowski S, & Leonardi R. (2015). Induction of Neuron-Specific Degradation of Coenzyme A Models Pantothenate Kinase-Associated Neurodegeneration by Reducing Motor Coordination in Mice. PLoS ONE, 10(6), e0130013.

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