The bloodstream forms of T. brucei were washed with PBS and resuspended and fixed in PBS containing 4% paraformaldehyde for 5 min at room temperature. The fixed parasites were washed with PBS and allowed to attach to coverslips for 5 min. The coverslips were submerged in PBS containing 0.1% Nonidet P40 for 5 min to permeabilize the cells. Subsequently, PBS containing 3% bovine serum albumin was added for a 1-h blocking period. After blocking, the cover slips were incubated for 1 h with a 1:1000 dilution of rabbit polyclonal anti-TbGRASP and 1 μg/mL of rat monoclonal anti-HA 3 F10 (Roche Diagnostics) in CanGetSignal Immunostain A (TOYOBO), followed by three 10-min washes in PBS containing 0.5% BSA. Following this, the cover slips were washed thrice in PBS containing 0.5% BSA and incubated for 1 h with a 1:1000 dilution of AlexaFluor 594-conjugated anti-rabbit IgG (Life Technologies) and 2 μg/mL of AlexaFluor 488-conjugated anti-rat IgG (Cell Signaling Technology, Danvers, MA) in CanGetSignal Immunostain B. The coverslips were subsequently washed thrice and mounted in antifade mounting solution containing DAPI (Vector Laboratories). Images were obtained with a confocal laser scanning microscope 510 (Zeiss). All images were obtained and processed under the similar settings.
Alexa fluor 488 conjugated anti rat igg
Manufactured by Cell Signaling Technology
Sourced in United States
Alexa Fluor 488-conjugated anti-rat IgG is a secondary antibody that recognizes and binds to rat immunoglobulin G (IgG) molecules. The antibody is labeled with the Alexa Fluor 488 fluorescent dye, which can be detected using a fluorescence microscope or flow cytometer.
Automatically generated - may contain errors
Lab products found in correlation
Ec800 cell analyzer, by Sony (3 mentions)
Alexa fluor 594 conjugated anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Rat monoclonal anti ha 3f10, by Roche (2 mentions)
Cangetsignal immunostain a, by Toyobo (2 mentions)
Antifade mounting solution containing dapi, by Vector Laboratories (2 mentions)
Triton x 100, by Solarbio (1 mentions)
Paraformaldehyde (pfa), by Solarbio (1 mentions)
Confocal laser scanning microscope, by Leica (1 mentions)
Mounting medium with dapi, by ZSGB-BIO (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Fujifilm (1 mentions)
Oregon green conjugated anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Fujifilm (1 mentions)
Alexa fluor 488 conjugated anti mouse igg, by Cell Signaling Technology (1 mentions)
8 protocols using alexa fluor 488 conjugated anti rat igg
1
Immunofluorescence Microscopy of T. brucei
2
Cell surface marker analysis
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The cells were harvested after a brief exposure to 0.25% trypsin/1 mM ethylenediaminetetraacetic acid (Nacalai Tesque, Inc.). After washing with 0.1% bovine serum albumin in phosphate-buffered saline, the cells were treated with primary mAbs (1 μg/mL) for 30 min at 4 °C and subsequently with Alexa Fluor 488-conjugated anti-rat IgG (1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA). Fluorescence data were obtained using an EC800 Cell Analyzer (Sony Biotechnology Inc.).
3
Cell Labeling and Analysis Protocol
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Cells were harvested after brief exposure to 0.25% trypsin/1 mM ethylenediaminetetraacetic acid (Nacalai Tesque, Inc.). After washing with 0.1% bovine serum albumin in PBS, the cells were treated with PMab-1 for 30 min at 4 °C, followed by treatment with Alexa Fluor 488-conjugated anti-rat IgG (1:1000; Cell Signaling Technology, Inc., Danvers, MA). Fluorescence data were acquired using the Cell Analyzer EC800 (Sony Corp., Tokyo, Japan).
4
Immunofluorescent Characterization of hESC-Derived Epithelial Cells
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hESCs were seeded on Matrigel-coated glass slides in 12-well plates and induced for 7 d to obtain hESC-Es, as described previously. hESC-Es were fixed in 4% paraformaldehyde (Solarbio, Beijing, China) for 15 min, permeabilized with 0.25% TritonX-100 (Solarbio, Beijing, China) for 15 min, blocked with 5% bovine serum albumin for 1 h at room temperature and then incubated simultaneously with primary antibodies, including monoclonal rabbit anti-P63 and polyclonal mouse anti-K18 (1:500, Cell Signaling Technology, Danvers, MA, USA), overnight at 4 °C. Cells were stained with fluorophore-conjugated secondary antibodies, including Alexa fluor 488-conjugated anti-rat IgG (Cell Signaling Technology, Danvers, MA, USA) and rhodamine (TRITC)-conjugated anti-mouse IgG (ZSGB-BIO, Beijing, China), at 1:500 and 1:100 dilutions, respectively for 1 h at room temperature. The cells were mounted using mounting medium with DAPI (ZSGB-BIO, Beijing, China). Immunofluorescent images were observed using a laser scanning confocal microscope (Leica, Japan) with LAS AF Lite software.
5
Cell Harvesting and Immunofluorescence Analysis
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Cell lines were harvested by brief exposure to 0.25% Trypsin/1 mM EDTA (Wako Pure Chemical Industries).(22 (link)) After washing with phosphate-buffered saline (PBS), the cells were treated with primary antibodies (1 μg/mL) for 30 min at 4°C, followed by treatment with Oregon green-conjugated anti-mouse IgG (Life Technologies), Alexa Fluor 488 conjugated anti-mouse IgG (Cell Signaling Technology, Danvers, MA), or Alexa Fluor 488 conjugated anti-rat IgG (Cell Signaling Technology). Fluorescence data were collected using a FACS Calibur flow cytometer (BD Biosciences, Braintree, MA) or a Cell Analyzer EC800 (Sony, Tokyo, Japan).
6
Immunofluorescence Microscopy of T. brucei
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The bloodstream forms of T. brucei were washed with PBS and resuspended and fixed in PBS containing 4% paraformaldehyde for 5 min at room temperature. The fixed parasites were washed with PBS and allowed to attach to coverslips for 5 min. The coverslips were submerged in PBS containing 0.1% Nonidet P40 for 5 min to permeabilize the cells. Subsequently, PBS containing 3% bovine serum albumin was added for a 1-h blocking period. After blocking, the cover slips were incubated for 1 h with a 1:1000 dilution of rabbit polyclonal anti-TbGRASP and 1 μg/mL of rat monoclonal anti-HA 3 F10 (Roche Diagnostics) in CanGetSignal Immunostain A (TOYOBO), followed by three 10-min washes in PBS containing 0.5% BSA. Following this, the cover slips were washed thrice in PBS containing 0.5% BSA and incubated for 1 h with a 1:1000 dilution of AlexaFluor 594-conjugated anti-rabbit IgG (Life Technologies) and 2 μg/mL of AlexaFluor 488-conjugated anti-rat IgG (Cell Signaling Technology, Danvers, MA) in CanGetSignal Immunostain B. The coverslips were subsequently washed thrice and mounted in antifade mounting solution containing DAPI (Vector Laboratories). Images were obtained with a confocal laser scanning microscope 510 (Zeiss). All images were obtained and processed under the similar settings.
7
Establishment of C3Mab Antibody Clones
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C3Mab-4 was established using the CBIS method together with C3Mab-3 [30 (link)]. C3Mab-7 was established using N-terminal peptide immunization as described previously [29 (link)]. An anti-mCCR3 mAb (clone J073E5) was purchased from BioLegend (San Diego, CA, USA). A secondary Alexa Fluor 488-conjugated anti-rat IgG was also purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).
8
Fluorescent Antibody Labeling Protocol
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Alexa-Fluor-488-conjugated anti-rat IgG was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).
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