For immunofluorescent analysis of CD34 expression, cells were grown on collagen I-coated coverslips placed on 6 well plates and reached a confluence of 60%–70% before the assay. Cells were fixed on coverslips with 4% paraformaldehyde in PBS for 15 min at room temperature (RT) and then permeabilized with 0.5% Triton X-100 for 20 min. After blockage in normal goat serum for 30 min at RT, cells were stained with primary CD34 antibody (1:200, Abcam, USA) at 4 °C for overnight in a wet box and then incubated with Cy3-conjugated goat anti-rabbit secondary antibody (1:100, Wuhan Boster, China) for 1 h at RT in dark. In the control cells, primary antibody was replaced with a mouse IgG isotype control (1:100, Sigma, USA). Coverslips were mounted to slides with ProLong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific, USA). Images were collected and analyzed using a fluorescence microscope (Olympus BX53, Olympus, Japan).
Mouse igg isotype control
Manufactured by Merck Group
Sourced in United States
The Mouse IgG isotype control is a laboratory reagent used as a negative control in immunoassays. It provides a non-specific binding reference to differentiate antigen-specific binding from non-specific binding.
Automatically generated - may contain errors
4 protocols using mouse igg isotype control
1
Immunofluorescent Analysis of CD34 Expression
2
Depletion of NK Cells and NKG2D During Desiccating Stress in Mice
3
Modulation of GEP in Human HCC Cells
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Human HCC cell lines Hep3B and HepG2 were purchased from American Type Culture Collection (Manassas, VA) and authenticated using short tandem repeat DNA profiles by the company. The cells were expanded and cultured immediately, or cryopreserved in multiple aliquots and used within 6 months of resuscitation as described.16 (link) Stable transfectants for GEP overexpression were established by transfecting GEP full-length cDNA (FL) into HepG2 cells, whereas GEP suppression was performed by transfecting GEP shRNA (sh) into Hep3B cells. Vector control and shRNA negative control were included as controls for transfections in HepG2 and Hep3B cells, respectively. All transfectants were maintained in advanced minimum essential media (AMEM) with 10% FBS and 0.4 mg/mL G418 (Life Technologies). GEP blockage in Hep3B was performed by incubating the cells with or without 50 μg/mL anti-GEP monoclonal antibody A23 (Versitech Ltd, Hong Kong) or mouse IgG isotype control (Sigma-Aldrich) for 24 h. Doses of A23 were previously titrated and 50 µg/mL of A23 alone did not exert any cytotoxic effect on HCC cells.21 (link)
4
Integrin α₅β₁ Expression in Glioblastoma Neurospheres
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The cell expression of integrin α5β1 in glioblastoma neurospheres was verified as previously described [18 (link)]. GBM-CCC-001, GBM-CCC-002 and GBM-CCC-003 neurospheres were collected into 15 mL Falcon tubes, pelleted down, and dissociated into single cells. After cell counting, 200,000 cells were transferred into Eppendorf tubes and incubated with the primary anti-integrin α5β1 antibody MAB1969 (Sigma-Aldrich, Burlington, MA, USA) or mouse IgG isotype control (Sigma-Aldrich, Burlington, MA, USA) at 1:100 dilution in PBSA (1% w/v bovine serum albumin (BSA) in phosphate buffer saline (PBS)) at 4 °C for 30 min. Following the incubation period, cells were pelleted and washed twice with ice-cold PBSA, then incubated with the FITC-conjugated anti-mouse IgG secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA) for 30 min at 4 °C Finally, cells were pelleted and washed twice with ice-cold PBSA, and flow cytometric analysis was performed immediately using BD FACSCanto (Integrated Imaging Center, Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD, USA). Cell autofluorescence was subtracted from all measurements.
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