A monolayer of HT-29 cells was infected as described above in “Cell adherence assay.” Following incubation, the monolayer was washed once with PBS and fixed in 4% paraformaldehyde for 15 minutes. The samples were subsequently incubated for 1 hour in PBS containing rabbit anti-UC34 (1:200), followed by an additional 1-hour incubation in PBS containing Alexa Fluor 488-conjugated goat anti-rabbit antibody (1:200; Abcam) and Hoechst 33342 (1:500; Thermo Fisher Scientific) (10 (link)). The samples were imaged using an LSM780 inverted confocal laser scanning microscope (Carl Zeiss) fitted with Plan Apochromat 40×/1.3-NA and 63×/1.4-NA oil objective lenses, with excitation at 405 nm and 488 nm. Tile scan images were stitched using Image Stitching plug-ins on Fiji (74 (link)). All images were processed using Imaris version 8.2.0 (Bitplane).
Alexa fluor 488 conjugated goat anti rabbit antibody
Manufactured by Abcam
Sourced in United Kingdom
Alexa Fluor 488 conjugated goat anti-rabbit antibody is a secondary antibody that binds to rabbit primary antibodies. It is conjugated to the Alexa Fluor 488 fluorescent dye, which has an excitation peak at 488 nm and an emission peak at 519 nm.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 800, by Zeiss (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Lsm780 inverted confocal laser scanning microscope, by Zeiss (1 mentions)
Clariostar plus microplate reader, by BMG Labtech (1 mentions)
Lsm 700 confocal laser scanning microscope, by Zeiss (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Rabbit anti tia1 antibody, by Abcam (1 mentions)
Blasticidin, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 conjugated goat anti mouse antibody, by Thermo Fisher Scientific (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Dapi solution, by Bioss Antibodies (1 mentions)
Image pro plus software, by Media Cybernetics (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
8 protocols using alexa fluor 488 conjugated goat anti rabbit antibody
1
Visualizing HT-29 cell adherence and infection
2
SARS-CoV-2 Pseudotype Binding Assay
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The effect of C1q or C4BP treatment on SARS-CoV-2 pseudotype binding to transfected A549 cells was evaluated using a cell-binding assay. Briefly, A549-hACE2+TMPRSS2 cells (20,000 cells/well) were seeded in a 96-well plate in a growth medium and left overnight at 37 °C. The next day, cells were challenged with SARS-CoV-2 lentiviral pseudoparticles treated with C1q, ghA, ghB, ghC, or C4BP and incubated in an incomplete growth medium for 2 h at 37 °C. This was followed by washing the plate three times with PBS; cells were then fixed for 1 min using 1% v/v paraformaldehyde (PFA) at RT. After washing three times with PBS, cells were incubated with rabbit anti-SARS-CoV-2 spike (1:200) polyclonal antibodies for 1 h at 37 °C and washed. Finally, the wells were probed with the Alexa Fluor 488 conjugated goat anti-rabbit antibody (1:200) (Abcam, Cambridge, UK) for 1 h at RT. The plate was read using a Clariostar Plus Microplate Reader (BMG Labtech, Cary, NC, USA).
3
Nrf2 Activation in HK-2 Cells
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HK-2 cell groups (control, RVS, CoCl2 and CoCl2+RVS) were grown on glass coverslips (n=5 per group). Distilled water and RVS were diluted in culture medium and applied to the cells for 24 h. CoCl2 was subsequently added to the culture medium for 24 h. Coverslips were fixed with 4% paraformaldehyde for 30 min at 4°C, blocked with 1% bovine serum albumin (Sigma-Aldrich; Merck KGaA) diluted in PBS for 1 h at 24°C and incubated with anti-Nrf2 rabbit polyclonal antibody (1:1,000, cat. no., sc-722; Santa Cruz Biotechnology, Dallas, TX, USA) for 24 h at 4°C. Subsequently, cells were incubated with Alexa Fluor® 488-conjugated goat anti-rabbit antibody (1:2,000, cat. no., ab150077; Abcam, Cambridge, UK) at room temperature for 1 h. Coverslips were then counterstained for 5 min with DAPI and mounted with ProLong Gold mounting media (cat. no., 8961; Cell Signaling Technology, Danvers, MA, USA) at 24°C and examined using the LSM 700 laser scanning confocal microscope (magnification, ×400; Zeiss AG, Oberkochen, Germany).
4
Immunofluorescent Imaging of Stress Granules
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The core components of SG, T-cell intracellular antigen-1 (TIA1), and GTPase-activating protein-binding protein 1 (G3BP1) are considered direct indicators of SG formation. Therefore, we used a double-labeled immunofluorescence (G3BP1 and TIA1) assay to observe SG generation in the cortex, PC12 cells, and primary cortical neurons. For immunofluorescent staining, frozen sections (10 μm thick) of the brain and cells (PC12 cells and primary cortical neurons) mounted on cover glass were fixed with 4% paraformaldehyde (PFA) for 20 min, permeabilized in 0.3% Triton X-100 solution for 10 min, blocked with blocking solution (5% BSA and 10% goat serum) for 1 h and finally incubated with primary antibodies (rabbit anti-TIA1 antibody, 1:1000, Abcam; mouse anti-G3BP1 antibody, 1:1000, Abcam, Cambridge, United Kingdom) and secondary antibodies (Alexa Fluor488-conjugated goat anti-rabbit antibody, 1:500, Abcam; Alexa Fluor555-conjugated goat anti-mouse antibody,1:500, Abcam) in the dark. DAPI staining for 10 min was used to label nuclei. Images were acquired using a confocal laser scanning microscope (LSM 800, Zeiss, Oberkochen, Germany). Each experiment was replicated three times and data are presented as the mean ± SEM.
5
Quantifying DC-SIGN Binding Interactions
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DC-SIGN-expressing HEK 293 (DC-HEK) cells, as reported by Lang et al. (31 (link)) were grown in DMEM-F12 (Life Technologies, UK) containing 10% v/v FCS and blasticidin (5 µg/mL) (Gibco). The cells were grown on 13 mm glass cover slips till a monolayer of cells was formed and then incubated with 15 µg/mL of recombinant ghA, ghB, and ghC (MBP as a negative control) separately in serum free medium and left to incubate for 30 min in 37°C. Cells were washed with PBS and fixed using 4% v/v paraformaldehyde for 10 min, rinsed again with PBS three times, and then blocked with 5% FCS for 30 min. The slides were incubated for 30 min with mouse anti-MBP antibody to detect MBP fusion proteins and rabbit anti-DC-SIGN antibody to reveal expression of DC-SIGN in DC-HEK cells. After three washes for 30 min each and incubation with secondary antibodies: Alexa Fluor 568 conjugated goat anti-mouse antibody (Thermo Fisher) and Alexa Fluor 488 conjugated goat anti-rabbit antibody (Abcam) for 30 min, the slides were then washed in PBS, mounted, and observed under Leica DM4000 Fluorescent microscope using Leica Application Suite.
6
Quantifying Mouse IVC Vascular Integrity
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Following euthanasia, mouse IVC was collected, fixed with 4% paraformaldehyde (overnight), frozen in Tissue-Tek® OCT medium and sliced. Blood vessel wall histomorphology and integrity was examined under a light microscope. Blood vessel wall ICAM-1, PECAM-1, tissue factor, and von Willebrand Factor (vWF) expression was measured using immunofluorescence microscopy (rabbit anti mouse ICAM-1, PECAM-1 and vWF antibodies were purchased from Abcam, and used at 1 µg/mL). For experimental control, no primary antibody but PBS was added to the sliced blood vessel samples. Secondary antibody was then added (Alexa Fluor 488-conjugated goat anti rabbit antibody, obtained from Abcam and used at 1 µg/mL), followed by immunofluorescence microscopy, to determine the fluorescence intensity of bound antibody. Image analysis was conducted using the Image J software. All intensity values were normalized to that of matching negative control.
7
Quantifying Stress Granule Formation in PC12 Cells
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PC12 cells were grown on glass coverslips in 24 well-plates at 1×105 cells per well. According to the experimental protocol, cells were transfected or co-transfected with miR-335 mimic, miR-335 inhibitor, small interfering (si)-RNA-ROCK2 and pLUC-ROCK2 plasmids, and then stimulated with serum-free medium for 24 h. Following fixation with 4% paraformaldehyde for 30 min at room temperature, PC12 cells were permeabilized with 0.3% Triton X-100 and blocked with 5% BSA. Then cells were incubated with diluted primary antibody (cat. no. 12133-2-AP; rabbit anti-TIA1 antibody; 1:1,000; ProteinTech Group, Inc.) at 4°C overnight. and incubated with the secondary antibody (cat. no. ab150077; Alexa Fluor488-conjugated goat anti-rabbit antibody; 1:5,000; Abcam) for 1 h in the dark. The PC12 cells were rinsed three times with PBS and stained with DAPI solution (BIOSS, Beijing, China) for nuclear labeling. The staining results were observed with a confocal laser scanning microscope (LSM 800; Carl Zeiss AG, Oberkochen, Germany). The formation of SGs was evaluated by calculating the percentage of positive cells with Image Pro Plus software (Version 6.0; Media Cybernetics, Inc.). Each experiment was replicated 3 times and the data are presented as the mean ± SEM.
8
SARS-CoV-2 Spike Pseudotype Binding Assay
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DC-HEK cells were cultured on 13 mm glass coverslips to form a monolayer, followed by incubation with SARS-CoV-2 spike pseudotypes (50µl) at 37°C. For 30 min, cells were rinsed with PBS and fixed using 1% w/v PFA for 1 min. The cells were washed three times with PBS, then blocked with 5% w/v BSA in PBS (Fisher Scientific) for 30 minutes. The cells were incubated for 30-min with mouse anti-human DC-SIGN antibodies to detect DC-SIGN and rabbit anti-SARS-CoV-2 Spike antibodies. Next, cells were washed and incubated with a staining buffer containing Alexa Fluor 647 conjugated goat anti-mouse antibody (Abcam), Alexa fluor 488 conjugated goat anti-rabbit antibody (Abcam), and Hoechst (Invitrogen, Life Technologies). This incubation was done in the dark for 45 min. After rinsing with PBS, the mounted coverslips were visualised under a Leica DM4000 microscope.
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