Immortalized HeLa cells were seeded on cover slips, at a density of 5 × 105 cells/well in standard 12‐well plates. The cells were 40%–50% confluent and then transfected with the recombinant plasmids pHAGE‐TBX5wt and pHAGE‐TBX5mut using Lipofectamine 2000 according to the manufacturer's instructions. At 48 h post‐transfection, the cells were fixed in 2% paraformaldehyde, permeabilized with 0.3% Triton X‐100, and incubated with a mouse anti‐TBX5 (1:100; Proteintech, USA) antibody at 4°C for 24 h and an Alexa488‐conjugated rabbit anti‐mouse antibody (1:200; Invitrogen, USA) at room temperature for 2 h. Immunofluorescence signals were visualized under a fluorescence microscope (Life Technologies). Photoshop CC 2019 was used to measure the cytoplasmic and nuclear fluorescence signals.
Alexa488 conjugated rabbit anti mouse antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Alexa488 conjugated rabbit anti-mouse antibody is a secondary antibody used in immunoassays and other laboratory techniques. It is designed to bind to mouse primary antibodies, allowing for the detection and visualization of target proteins or antigens.
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Lab products found in correlation
Fluorescence microscope, by Thermo Fisher Scientific (1 mentions)
Mouse anti ha antibody, by Merck Group (1 mentions)
Facscan, by BD (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Mcf 10a, by American Type Culture Collection (1 mentions)
Cholera toxin, by Merck Group (1 mentions)
Epidermal growth factor (egf), by Merck Group (1 mentions)
Horse serum, by Thermo Fisher Scientific (1 mentions)
Insulin, by Thermo Fisher Scientific (1 mentions)
Hydrocortisone, by Merck Group (1 mentions)
Growth factor reduced matrigel, by Thermo Fisher Scientific (1 mentions)
Gefitinib, by Selleck Chemicals (1 mentions)
Doxorubicin, by Selleck Chemicals (1 mentions)
Mouse anti β actin, by Merck Group (1 mentions)
Mouse anti α tubulin, by Merck Group (1 mentions)
Afatinib, by Selleck Chemicals (1 mentions)
Carl epifluorescence microscope, by Zeiss (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
4 protocols using alexa488 conjugated rabbit anti mouse antibody
1
Quantifying TBX5 Localization in Transfected HeLa Cells
2
Cell Surface Expression Analysis of Receptor Chimeras
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Determination of the receptor's cell surface expression was performed as previously described by Müller et al. [31] (link). For permeabilized cell assay, in the first step cells were fixed with 1% paraformaldehyde for 10 min on ice following incubation with PBS containing 0.1% bovine serum albumin and 0.2% saponin for 30 min. Saponin was supplemented in all subsequent buffers. Subsequently, cells were incubated for 1 h with a 1∶400 dilution of a mouse anti-HA antibody (Sigma). Cells were washed twice and incubated for 1 h with a 1∶400 dilution of an Alexa488 conjugated rabbit anti-mouse antibody (Invitrogen). Before FACS analysis (FACscan; BD Biosciences), cells were washed twice and fixed with 1% paraformaldehyde. Receptor expression was determined by fluorescence intensity; the percentage of signal positive cells corresponded to transfection efficiency.
The cell surface expression of all characterized chimeras were normalized to the wild type receptor containing the respective transmembrane domain.
The cell surface expression of all characterized chimeras were normalized to the wild type receptor containing the respective transmembrane domain.
3
Comparative Analysis of MCF10 Breast Cell Lines
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MCF10A (ATCC, Manassas, VA) and MCF10CA1a (gift from Pauley Robert from the Barbara Ann Karmanos Cancer Institute, Detroit, Michigan) cells were maintained in DMEM/F12 (Invitrogen, Carlsbad, CA) supplemented with 5% horse serum (Invitrogen), 500ng/ml hydrocortisone (Sigma-Aldrich, St. Louis, MO), 100ng/ml cholera toxin (Sigma-Aldrich), 10 μg/ml insulin (Invitrogen) and 20ng/ml epidermal growth factor (EGF, Sigma-Aldrich) (“full media”). Some assays were performed in reduced (2%) serum media in the presence or absence of EGF. Gefitinib, afatinib, docetaxol and doxorubicin were purchased from Selleck Chemicals (Houston, TX) and maintained as a 10 mM stock in dimethyl sulphoxide (DMSO). Mouse anti-β-actin, mouse anti-α-tubulin and rabbit anti-mouse HRP-conjugate antibodies were obtained from Sigma-Aldrich, mouse anti-EGFR monoclonal antibody (clone 225) was isolated from hybridoma cultures (HB-8508 from the ATCC) as previously described [35 (link)], Alexa488 conjugated rabbit anti-mouse antibody was obtained from Invitrogen, and IRDye 800CW goat anti-mouse antibody was obtained from Millenium Science (Mulgrave, Australia). Growth factor-reduced Matrigel was obtained from Invitrogen.
4
Immunolocalization of PGRMC in Taenia solium
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Cultured T. solium cysticerci were washed with PBS 1× and embedded in Tissue Tek (Triangle Biomedical Science, Arizona, USA), and immediately frozen at -80 °C until use. Parasitic tissue sections (5 μm) were fixed with frozen acetone for 10 min, washed 3 times in PBS-Tween 0.3% and blocked for 1 h with PBS containing 1% bovine albumin. Cross-sections were then incubated with a 1:100 dilution of PGRMC polyclonal antibody obtained from T. spiralis PGRMC (cloned, sequenced, synthesized and produced by Dr. Romel Hernández-Bello, who kindly donated it to us) for 45 min at 37 °C, washed with PBS and then incubated with Alexa 488-conjugated rabbit anti-mouse antibody (Invitrogen, California, USA) at 1:300 dilution. Control experiments were assessed incubating the 5 μm thick tissue sections in the presence of Alexa 488-conjugated rabbit anti-mouse antibody alone at the same dilution. To eliminate background fluorescence, samples were contrasted with 0.025% Evans Blue for 10 min. After two single washings, samples were mounted in Vectashield mounting medium (Vector Laboratories Inc.,Boston, USA) and examined with a Carl Zeiss epifluorescence microscope (Carl Zeiss, Berlin, Germany).
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