For immunofluorescence (IF) assays, cells were counted and seeded at 5×104 cells on sterile glass coverslips. For assessing cilia, MEFs were serum-starved for 48 hours prior to analysis. Where indicated, drugs were added 24 hours prior to fixation. Cells were fixed in 4% PFA (Sigma Aldrich, St Louis, USA) or ice-cold Methanol (100%) for the centrosomal protein in PBS for 20 minutes at RT and permeabilized in 0.1% TritonX-100 (Sigma Aldrich) for 10 minutes or 90 seconds (cilia experiments) and blocked in 3% or 1% (cilia experiments) bovine serum albumin (BSA; Sigma Aldrich) in PBS for 1 hour in a humidified chamber at RT. Coverslips were washed and incubated with Alexa-fluor-conjugated secondary antibodies (Sigma Aldrich) diluted in 3 or 1% BSA (1:1000) for 30 minutes at 37°C in a humidified chamber in the dark. Coverslips were mounted using Prolong gold anti-fade mounting medium (Life Technologies). Imaging was performed on a DeltaVision personal DV deconvolution microscope and DeltaVision Ultra (super-resolution) (Applied Precision, GE Healthcare, Issaquah, WA) and analyzed using the GE DeltaVision software package. Automated counting was performed using script modules of Fiji-ImageJ software (Java3D, Minnesota, USA).
Alexa fluor conjugated secondary antibodies
Manufactured by Merck Group
Alexa Fluor-conjugated secondary antibodies are fluorescent-labeled antibodies used in various immunodetection techniques. They bind to primary antibodies and amplify the detection signal, enabling visualization and quantification of target molecules.
Automatically generated - may contain errors
Lab products found in correlation
Prolong gold antifade mounting medium, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Deltavision ultra, by Cytiva (1 mentions)
Mz10f, by Leica (1 mentions)
Lsm 510 confocal microscope, by Zeiss (1 mentions)
Alexa flour 488, by Abcam (1 mentions)
Ab28364, by Abcam (1 mentions)
Qimaging camera, by Olympus (1 mentions)
Anti caspase 1 p20, by Santa Cruz Biotechnology (1 mentions)
Mab1420, by R&D Systems (1 mentions)
Anti il 1β, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Hoechst, by Merck Group (1 mentions)
C fos, by Cell Signaling Technology (1 mentions)
βiii tubulin tuj1, by BioLegend (1 mentions)
5 protocols using alexa fluor conjugated secondary antibodies
1
Immunofluorescence Assay for Cilia Analysis
2
Immunolabeling of Embryonic Patterning Proteins
3
Immunofluorescence Imaging of Transfected HEK293 Cells
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HEK293 cells were transfected with different plasmid DNAs in the indicated groups. A total of 2.5×10
4 cells/mL were cultured on slips at 37°C to 50%–60% confluence. The cells were exposed to 4% paraformaldehyde at a temperature of 4°C for 30 min and subsequently incubated in a blocking solution consisting of PBS with 10% (w/v) normal goat serum for 30 min at room temperature. Fixed cells were incubated with primary antibodies diluted in PBS with 1% BSA overnight at 4°C. Detection of the primary antibodies was performed using Alexa Fluor-conjugated secondary antibodies (Sigma-Aldrich). Confocal microscopy was employed to record images with a Zeiss LSM 510 confocal microscope (Carl Zeiss, Oberkochen, Germany). All images were then analysed using ImageJ (NIH, Bethesda, USA).
4 cells/mL were cultured on slips at 37°C to 50%–60% confluence. The cells were exposed to 4% paraformaldehyde at a temperature of 4°C for 30 min and subsequently incubated in a blocking solution consisting of PBS with 10% (w/v) normal goat serum for 30 min at room temperature. Fixed cells were incubated with primary antibodies diluted in PBS with 1% BSA overnight at 4°C. Detection of the primary antibodies was performed using Alexa Fluor-conjugated secondary antibodies (Sigma-Aldrich). Confocal microscopy was employed to record images with a Zeiss LSM 510 confocal microscope (Carl Zeiss, Oberkochen, Germany). All images were then analysed using ImageJ (NIH, Bethesda, USA).
4
Immunofluorescence Assay of Wound Skin Tissues
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For immunofluorescence assay, sections of paraffin-embedded wound skin tissues from WT or Mfge8−/− mice were deparaffinized and subjected to antigen retrieval. After permeabilized with 0.03% Triton X-100, and blocked in 5% BSA, anti-IL-1β (Abcam), anti-caspase-1 p20 (active caspase-1, Santa Cruz #SC-1218, Cell Signaling Technology, MA, USA), anti-CD31 (Abcam ab28364), and anti-α-smooth muscle actin (α-SMA) antibodies (R&D Systems MAB1420) were incubated overnight at 4°C. Secondary Alexa Flour 488 or Alexa Flour 647-conjugated antibodies (Abcam) were added for 2 h and then stained with DAPI (Sigma-Aldrich). For NETs assay, skin tissues sections were stained using anti-CitrH3 and anti-MPO antibodies (Abcam), followed by Alexa Fluor-conjugated secondary antibodies, and DAPI (Sigma-Aldrich). For P2X7 receptor detection, skin tissues were incubated with anti-P2X7 receptor antibody (Abcam ab195356), and following with Alexa Flour 647-conjugated antibodies. Sections (×200 or ×400) were imaged with Olympus BX51 microscope and Qimaging camera, and more than five fields per section were obtained.
5
Immunocytochemistry Protocol for Cell Labeling
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For immunocytochemistry (ICC), cells were fixed with 4% paraformaldehyde (PFA) 15 min on ice, washed and incubated in blocking solution (1% Bovine serum albumin (BSA), 0,25% Triton X-100 (0,25% Tx) in 0.1M PBS) for 1 h at RT. Primary antibody (βIII tubulin (Tuj1, 1:1000, BioLegend), c-Fos (1:200, Cell Signaling), ChR2 (1:500, Progen), mCherry (1:500, Abcam) GFP (1:500, ThermoFisher Scientific) was added to the cells in blocking solution and incubated O/N at 4 °C. After washing, the cells were incubated 1 h at RT with Alexa-Fluor-conjugated secondary antibodies (568 goat and 488 goat and donkey) and Hoechst (Sigma Aldrich).
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