Cells were fixed with 3.75% paraformaldehyde in PBS for 20 min, permeabilized with 0.3% Triton X‐100 for 10 min, blocked with 1% bovine gelatin in PBS for 10 min three times, and then washed with PBS for 5 min three times. Mitochondria were stained using a 1:100‐diluted rabbit anti‐TOM20 primary antibody (Santa Cruz). Cells were incubated with the antibody in a wet chamber for 2 h at room temperature, blocked three times with gelatin in PBS for 10 min, and washed three times with PBS for 5 min. Cells were then incubated with a 1:100‐diluted AlexaFluor 594‐conjugated anti‐rabbit secondary antibody (ThermoFisher) in a wet chamber for 45 min at room temperature, rinsed with gelatin in PBS for three times, then nuclei were stained by incubating the cells with 1 μg/ml Hoechst 33342 in PBS for 10 min. Finally, the cells were washed three times with PBS for 5 min and mounted on glass slides before being analyzed by a Zeiss sp5 confocal microscope.
Alexa fluor 594 conjugated anti rabbit secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 594-conjugated anti-rabbit secondary antibody is a laboratory reagent used for detection in immunoassays. It is a highly specific and sensitive antibody that binds to rabbit primary antibodies, allowing visualization of target proteins or other biomolecules in a sample. The antibody is conjugated to the Alexa Fluor 594 fluorescent dye, enabling detection using appropriate instrumentation.
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Lab products found in correlation
Alexa fluor 488 conjugated anti mouse secondary antibody, by Thermo Fisher Scientific (3 mentions)
Lsm 510 meta, by Zeiss (2 mentions)
Anti rabbit cd31 primary antibody, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Alexafluor488 conjugated anti chicken secondary antibody, by Thermo Fisher Scientific (2 mentions)
Chicken anti gfp, by Thermo Fisher Scientific (2 mentions)
8 champer slides, by Thermo Fisher Scientific (2 mentions)
Dmi8 microscope, by Leica camera (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (2 mentions)
Sp5 confocal microscope, by Zeiss (1 mentions)
Prolong gold antifade reagent, by Zeiss (1 mentions)
Bx61wi, by Olympus (1 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Polarstar omega microplate reader, by BMG Labtech (1 mentions)
Lsm 510 confocal microscope, by Zeiss (1 mentions)
Leica fluorescent microscope, by Leica Microsystems (1 mentions)
Formaldehyde, by Ted Pella (1 mentions)
Permanox chamber slide, by Thermo Fisher Scientific (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
20 protocols using alexa fluor 594 conjugated anti rabbit secondary antibody
1
Mitochondrial Localization Immunofluorescence Protocol
2
Analyzing Autophagy in Fluorescently Labeled Cells
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Cells were plated onto poly-L-lysine coated round coverslips in a 6-well plate, grown overnight and subjected to experimental conditions as indicated. Stable cell lines expressing recombinant fluorescence proteins were imaged immediately after mounting on glass slides with ProLong™ Gold Antifade Mountant with DAPI (Thermo). For immunofluorescence, cells were fixed with 4% paraformaldehyde, briefly washed with PBS, and subsequently permeabilized with 0.5% Triton X-100 in PBS for 10 min followed by 1 h blocking in 2% BSA at RT. Coverslips were then incubated with anti-LC3A/B antibody (4108 S, CST; 1:250) for overnight at 4 °C, washed three times with PBS, and incubated again with DAPI and Alexafluor™ 594 conjugated anti-rabbit secondary antibody (A11012, Thermo; 1:250) for 1 h at RT. After 3 washes with PBS, coverslips were mounted on glass slides with ProLong™ Gold antifade reagent and examined under LSM510 META (Carl Zeiss) and BX61WI (Olympus) confocal microscopes. LC3-specific puncta were counted manually by ImageJ in minimum 25 cells per experimental group.
3
Quantification of BLF Adhesion to Cells
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Adhesion of native (bLF) and deglycosylated bLF (bLFdeg) to the cell surface was quantified by an adaptation of the fluorescence activation cell sorting (FACS) method (Greenfield, 2022 (link)). HeLa and Caco2 cells were cultured in black 96‐well plates at a density of 3.5 × 104 and 5 × 104 cells per well, respectively, and were incubated at 37°C in 5% CO2, HeLa was incubated for 24 h and Caco2 for 48 h. Then, cell layers were washed with PBS and they were incubated with 10 μM bLF and bLFdeg in DMEM, without antibiotics or supplements, for 10 min and 1 h at 37°C. Control wells without bLF or bLFdeg were also included. Cells were washed three times with PBS, fixed with 100 μL of 4% PFA for 10 min at room temperature in the dark, washed three times, and incubated with 50 μL of blocking solution (4% BSA in PBS) for 1 h at room temperature. After that, cells were treated with 50 μL of rabbit anti‐LF primary antibody (Sigma Aldrich) at 1:400 in 1% BSA in PBS. Then samples were washed three times with PBS and after this, they were incubated with 50 μL of Alexa Fluor 594‐conjugated anti‐rabbit secondary antibody (Thermofisher Scientific) at 1:500 in 1% BSA in PBS for 1 h at room temperature. After that, they were washed three times with PBS and 20 μL of PBS was added for the measurement of fluorescence at 540/610 nm using a POLARstar Omega microplate reader (BMG LABTECH).
4
Immunoblotting and Fluorescence Microscopy
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To further substantiate the findings of immunoblotting, all cells were re-plated on fibronectin-coated coverslips and cultured for 24 h. They were then fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. Coverslips were washed with PBS and then incubated with specific first antibodies respectively, followed by Alexa Fluor 594-conjugated anti-rabbit secondary antibody (Thermo Scientific, #A11012) labeling. The cells were then reacted with DAPI for nuclear staining. For live cell labeling and recycling experiment, RG108-treated PANC-1 cells were labeled with mouse antibody against Ecad (CST, #5296) on ice and subjected immediately to calcium chelation by addition of 0.02% EDTA. They were then restored back to complete medium at 37 ℃ and 5% CO2 environment. Once anchored on coverslip, cells were fixed at different time points and probed for INPP4B with rabbit antibody (CST, #14,543), followed by Alexa Fluor 488 and 594-conjugated secondary antibodies (CST, #4412, #8890) labeling. All fluorescence images were acquired under an alpha Plan-Fluarobjective lens of Zeiss LSM 510 confocal microscope.
5
Biotin-Induced ABCG2 Trafficking in HeLa Cells
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HeLa cells, previously seeded onto an eight-well chamber at 2 × 104 cells/well density and grown for 24 h, were transfected with the RUSH-ABCG2 variants as described above. Twenty-four hours after transfection, the cells were subjected to 100 μM biotin for the indicated times (or remained untreated–0 h). Following this, the samples were gently washed with PBS and then fixed with 4% PFA for 10 min at room temperature. After several washing steps, the cells were blocked for 1 h at room temperature in Dulbecco’s modified phosphate buffer saline (DPBS) containing 2% bovine serum albumin, 1% fish gelatine, 0.1% Triton X-100, and 5% goat serum (blocking buffer). Next, the samples were incubated with anti-Giantin primary antibody (1:1000 in blocking buffer, BioLegend, cat. 924302) at 4°C overnight. After being washed with PBS, the cells were incubated with Alexa Fluor-594 conjugated anti-rabbit secondary antibody (1:250 in blocking buffer, Thermo Fisher Scientific, cat. A11012) for 1 h at room temperature.
6
Immunofluorescence Staining of Cell Markers
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Cells grown on glass coverslips were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Then, the cells were blocked with 2% bovine serum albumin (BSA) and incubated with anti-Brdu, anti-YAP, anti-E-cadherin, or anti-β-catenin antibodies overnight at 4 °C. After three washes with phosphate-buffered saline (PBS), the cells were incubated with Alexa Fluor 488-conjugated anti-mouse secondary antibody or Alexa Fluor 594-conjugated anti-rabbit secondary antibody (Thermo Fisher Scientific, NY, USA) for 1 h at room temperature. DAPI (4ʹ,6-diamidino-2-phenylindole) was used to stain the cell nuclei. Images were captured using a Leica fluorescent microscope (Leica Microsystems, Inc., New York, NY, USA).
7
Immunofluorescence Analysis of Stem Cell Differentiation
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To evaluate differentiation of stem cells and endothelial cells, they were fixed with 10% NBF for 1 h, blocked with 2% bovine serum albumin (BSA; Sigma‐Aldrich) for 2 h, and permeabilized with 2% Triton X‐100 for 2 h. The samples were incubated with an anti‐mouse osteopontin (OPN) primary antibody (5 μg/ml; Invitrogen, USA) and an anti‐rabbit CD31 primary antibody (5 μg/ml; Invitrogen, USA) overnight at 4°C. The primary antibody‐treated samples were stained with Alexa Fluor 488‐conjugated anti‐mouse secondary antibody (1:50 in DPBS; Invitrogen, USA) or Alexa Fluor 594‐conjugated anti‐rabbit secondary antibody (1:50 in DPBS; Invitrogen, USA) for 1 h, and counterstained with DAPI (5 μM in DPBS). The confocal microscope was used to visualize the stained cells. OPN‐ and CD31‐positive areas were quantified using ImageJ software. All values are expressed as means ± SD (n = 4).
8
Immunofluorescence Staining of TOM70 in HeLa Cells
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HeLa cells grown on Permanox chamber slides (Thermo Scientific, 177437) were fixed in 4% formaldehyde (Ted Pella, Redding, CA, USA, 18505) diluted in phosphate buffered solution (PBS). Cells were then permeabilized in PBS with 0.25% Triton-X 100 for 10 min, washed in PBS and blocked for one hour in blocking solution (PBS + 0.1% Tween-20 + 1% bovine serum albumin). Cells were then incubated overnight at 4 °C in anti-TOM70 antibody (1:200, Proteintech, Rosemont, IL, USA, 14528-1-AP) diluted in blocking solution. Cells were then washed in PBS and incubated for one hour protected from light in Alexa Fluor 594-conjugated anti-rabbit secondary antibody (1:100, Invitrogen, Carlsbad, CA, USA, A11037) diluted in blocking solution. Cells were then washed, and coverslipped in Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA, H-1400).
9
Visualizing YscF Protein Localization
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Visualization of YscF was carried out as previously described (31 (link)), with the following modifications. Coverslips were blocked overnight with phosphate-buffered saline with Tween 20 (PBST) plus 3% bovine serum albumin (PBST-BSA) at 4°C. The blocking solution was removed, an anti-YscF primary antibody was added at 1:10,000 in PBST-BSA, and the mixture was rocked at 4°C for 4 h. The coverslips were carefully rinsed in ice-cold PBST plus 0.1% Tween 20 several times, incubated with an Alexa Fluor 594-conjugated anti-rabbit secondary antibody (Invitrogen) at 1:10,000 in PBST-BSA, rocked at 4°C for 3 h, and rinsed again in ice-cold PBST plus 0.1% Tween 20 several times. The coverslips were then stained for nuclear material with Hoechst 33342 (Thermo Scientific) at 1:10,000 in PBST-BSA and left in the dark at room temperature for 30 min. The coverslips were washed in ice-cold PBST plus 0.1% Tween 20 several times, allowed to dry briefly, mounted onto glass coverslips with ProLong Gold (Thermo Scientific), and sealed with clear nail polish. Images were taken with a Zeiss Axio Imager Z2 widefield microscope under 63×/1.4 oil immersion with Zen software and pseudocolored and merged in FIJI. The conditions were blinded, and the puncta associated with a single bacterium were counted.
10
Localization of Endogenous Humanin in Rat Testes
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