To label live human pancreatic β-cells for fluorescence-activated cell sorting (FACS) and imaging, slices or dissociated cells incubated in DMEM + 2% FBS containing antibodies (Sigma-Aldrich, 1:100 dilution) against pan-endocrine marker HPi2 (HIC1-2B4) and non-β endocrine cell marker HPa3 (HIC3-2D12) for 30 min at 4 °C. After washing with cold DMEM, cells or slices were transferred to DMEM + 2% FBS containing pre-adsorbed Alexa Fluor conjugated secondary antibody (Abcam, 1:200) for 30 min at 4 °C. To minimize background staining, 5% goat serum were used for blocking the secondary antibody. Primary antibody against ANTI-HPI2, CLONE HIC1-2B4 (Sigma-Aldrich, MABS1999-100UG, 1:500) and ANTI-HPA3, CLONE HIC3-2D12 (Sigma-Aldrich, MABS1998, 1:500) were obtained from Millipore Sigma. Secondary antibody, Goat Anti-Mouse IgM mu chain (DyLight® 550) (Abcam, ab98675, 1:1500) and Goat Anti-Mouse IgG2b heavy chain (PE/Cy7 ®) (ab130790, 1:1500) were purchased from Abcam.
Alexa fluor conjugated secondary antibody
Manufactured by Abcam
Sourced in United Kingdom, United States
Alexa Fluor-conjugated secondary antibodies are fluorescent-labeled antibodies used to detect and visualize target proteins in various experimental techniques, such as immunofluorescence, flow cytometry, and western blotting. They provide a sensitive and specific way to amplify and detect the signal from primary antibodies bound to target proteins.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (2 mentions)
Hyaluronidase, by Merck Group (2 mentions)
Pepsin, by Merck Group (2 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Goat serum, by R&D Systems (1 mentions)
Porcine stomach mucin, by Merck Group (1 mentions)
Fitc labeled phalloidin, by Merck Group (1 mentions)
Bafilomycin a1, by Solarbio (1 mentions)
Alamar blue assay, by Solarbio (1 mentions)
2 n n dimethylamino ethyl methacrylate dmaema, by Thermo Fisher Scientific (1 mentions)
β propiolactone, by J&K Scientific (1 mentions)
Piercetm coomassie plus bradford assay kit, by Thermo Fisher Scientific (1 mentions)
Streptozotocin (stz), by Merck Group (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Solarbio (1 mentions)
Triton x 100, by MP Biomedicals (1 mentions)
34 protocols using alexa fluor conjugated secondary antibody
1
Labeling Pancreatic β-Cells for FACS and Imaging
2
Analyzing IL-6 Expression in LPS-Stimulated Cells
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Cells were cultured on 8-well glass chamber slides (Millicell® EZ SLIDE 8-well glass, sterile, Catalog No. PEZGS0816, Millipore, USA) in the same manner as described for the experiments performed in tissue culture plates. Cells were pre-treated with 9 mM MSO for one hour prior to the addition of 1 μg/kg LPS. Cells treated with LPS alone served as positive controls, whereas cells treated with MSO alone or left untreated served as negative controls. After 6 h of LPS stimulation, the medium was aspirated, and cells were washed three times with PBS before being fixed to the chamber slides with a 4% paraformaldehyde solution in PBS for 10 min. Cells were made permeable by the addition of a 0.1% Triton X-100 solution in PBS for 20 min, followed by an overnight incubation with a monoclonal antibody specific for mouse IL-6 (Cell signaling technology, USA, Catalog No. D5W4V) at 4 degrees. Cells were washed before incubation with an Alexa Fluor-conjugated secondary antibody (Abcam, USA, Catalog No. ab15007) in the presence of 10% goat serum (R&D systems, USA). A mounting medium containing the nuclear stain DAPI (Electron Microscopy Sciences, USA, Catalog No. 17985–50) was applied to a cover slip, and fluorescence was visualized under a confocal microscope.
3
Insulin-Loaded Mucoadhesive Nanoparticles
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2-(N,N'-dimethylamino) ethyl methacrylate (DMAEMA, 98%) was purchased from Alfa Aesar. β-Propiolactone (98%) and 2-cyanoprop-2-yl-dithiobenzoate were from J&K Scientific Ltd. Porcine insulin, FITC-labeled insulin and trypsin were purchased from Dalian Meilun Biotechnology Co., Ltd. 2,2'-Dicyano-2,2'-azopropane (AIBN) was from Aladdin Industrial Corporation. MTT, FITC-labeled phalloidin, porcine stomach mucin and STZ were obtained from Sigma Aldrich. LysoTracker deep and PierceTM Coomassie Plus (Bradford) Assay Kit were purchased from Thermo Fisher Scientific. Alamar blue assay, 4% paraformaldehyde, normal goat serum and bafilomycin A1 were from Beijing Solarbio Science & Technology Co., Ltd. The Eudragit L 100-55 was from Shanghai Chineway Pharmaceutical Technology Co., Ltd. Triton® X-100 was from MP Biomedicals, LLC. Anti-claudin-4 antibody and Alexa-Fluor-conjugated secondary antibody were obtained from Abcam. Anti-integrin αV and β1 antibodies were purchased from Affinity Biosciences LTD. All plates used in cellular experiments were Corning Incorporated.
4
Cytoskeletal Dynamics Visualization in HCT116 Cells
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HCT116
cells were seeded on coverslips precoated with 0.1% gelatin in six-well
culture plates at a density of 1 × 105 cells/well.
The following day, the cells were treated with the tested compounds
at concentrations corresponding to IC50 for 24 h. Following
the treatment, the cells were washed with PBS, fixed with 4% p-formaldehyde,
washed, permeabilized with 0.1% Triton X-100, and blocked. Samples
for actin staining were blocked with 1.5% BSA for 1 h and then stained
with Alexa Fluor 488-conjugated Phalloidin (Thermo Fisher Scientific,
1:50 dilution, 20 min). Samples for tubulin staining were blocked
with 5% goat serum for 1 h and incubated with primary antibody (anti-α-tubulin,
Abcam, 1:200 dilution, 1 h) and Alexa Fluor-conjugated secondary antibody
(goat antirabbit, Abcam, 1:500 dilution, 1 h). Both groups of samples
were mounted with ProLong Diamond Antifade Mountant with DAPI (Invitrogen).
Cells were visualized on a confocal microscope Leica TCS SP8 SMD.
cells were seeded on coverslips precoated with 0.1% gelatin in six-well
culture plates at a density of 1 × 105 cells/well.
The following day, the cells were treated with the tested compounds
at concentrations corresponding to IC50 for 24 h. Following
the treatment, the cells were washed with PBS, fixed with 4% p-formaldehyde,
washed, permeabilized with 0.1% Triton X-100, and blocked. Samples
for actin staining were blocked with 1.5% BSA for 1 h and then stained
with Alexa Fluor 488-conjugated Phalloidin (Thermo Fisher Scientific,
1:50 dilution, 20 min). Samples for tubulin staining were blocked
with 5% goat serum for 1 h and incubated with primary antibody (anti-α-tubulin,
Abcam, 1:200 dilution, 1 h) and Alexa Fluor-conjugated secondary antibody
(goat antirabbit, Abcam, 1:500 dilution, 1 h). Both groups of samples
were mounted with ProLong Diamond Antifade Mountant with DAPI (Invitrogen).
Cells were visualized on a confocal microscope Leica TCS SP8 SMD.
5
Collagen I/III and α-SMA Protein Expression
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Regarding the abundance of collagens I and III expression, aliquots of culture media were collected and 5 µg of protein were extracted from fibroblasts to evaluate α-SMA expression. Proteins were separated using NuPAGE® 4 to 12% Bis-Tris gels (Novex, Paisley, United Kingdom) and transferred onto nitrocellulose membranes. After blocking with Odyssey blocking buffer (LI-COR Biosciences, Cambridge, United Kingdom), the membranes were incubated with antibodies against human collagen type I or III (1/1000, Abcam), α-SMA (1/200, Abcam), and β-actin (1/1000, Abcam) at 4°C overnight. Then, membranes were washed with tris buffered saline with Tween (TBS-T) and incubated with an Alexa Fluor–conjugated secondary antibody (1/1000, Abcam) at 4°C for 1 hour. Immunoreactive bands were visualized with an Odyssey Infrared Scanner (LI-COR Biosciences). The intensity of each band was determined using ImageJ software (version 1.48v; National Institutes of Health, Bethesda, MD). The experiment was performed three independent times.
6
Tyloxapol Regulates p65 Translocation
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The effect of tyloxapol on p65 nuclear translocation was observed by immunofluoresistation. After 30-min stimulation using osteoclast differentiation medium with or without tyloxapol, raw 264.7 cells were immersed in the solution containing anti-p65 antibody for 3 h, and incubated with Alexa Fluor® conjugated secondary antibody (abcam) for 15 min. Finally, DAPI was used to incubate the cells for 10 min to visualize the nucleus. Immunofluorescence images were obtained under a confocal microscope.
7
Quantifying Autophagy in AML12 Cells
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AML12 cells were fixed in 10% formalin for 10 min and then permeabilized with 0.1% Triton-X100 for 15 min. The cells were imaged using a confocal microscope after incubation with anti-LC3 (1:500) and a Alexa Fluor conjugated secondary antibody (Abcam, Cambridge, UK), (Carl Zeiss, Oberkochen, Germany).
8
Immunofluorescence Detection of Inflammasome Proteins
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Immunofluorescence staining was performed to detect the expression of NLRP3/Caspase-1/GSDMD in macrophages. Briefly, the histologic sections were fixed with cold acetone for 10 min, penetrated by 0.3% Triton X-100 for 15 min, and then blocked with goat serum. Subsequently, the sections were incubated with the antibody at 4 °C overnight, followed by incubation with Alexa Fluor-conjugated secondary antibody (Abcam, USA) in the dark for 1 h. The nuclei were stained by 4′,6-diamidino-2-phenylindole (DAPI; Beyotime, China) for 5 min. The sections were imaged under fluorescence microscope.
9
Immunofluorescence Staining of Spinal Cord and Brain Endothelial Cells
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Spinal cord tissues were fixed in 4% paraformaldehyde (PFA) dissolved in PBS for 24 h. Next, the samples were embedded in paraffin and then cut into 5-µm-thick slices. The slices were successively deparaffinized and rehydrated. For immunofluorescence staining in HBMECs, cells’ climbing slices were also fixed with 4% PFA for 15 min. After washing with PBS, the tissue or cell slices were blocked with 5% BSA dissolved in PBS at 37°C for 30 min. Subsequently, the slices were incubated with the following antibodies at 4°C overnight: ZO-1 (1:250), β-catenin (1:250), LC3 (1:500), and CD31 (1:500; Santa Cruz). Then, the slices were further treated with the corresponding Alexa Fluor conjugated secondary antibody (1:1,000; Abcam) for 1 h at 37°C. DAPI was used for nuclei staining. Fluorescence intensity in cells was imaged using a Leica microscope.
10
Immunofluorescence Analysis of Murine Tibia
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Immunofluorescence analysis of the frozen sections of the tibia was performed as previously described. The following primary antibodies used were Ki67 (Millipore, Burlington, MA, USA), anti-Col X (Abclonal, A11645, 1:100), anti-phosphatase-ERK1/2 (Cell Signaling Technology, 4370, 1:100), anti-phosphatase-STAT1 (Cell Signaling Technology, 9172, 1:100), anti-SOX9 (Abcam, ab185966, 1:100), anti-Col2α1 (Thermo Fisher, PA1-26206, 1:100), and anti-aggrecan (Thermo Fisher, MA3-16,888, 1:100). Alexa Fluor 594 concanavalin A (Invitrogen) was used for colocalization with collagen. For Col X antigen retrieval, the sections were digested with 2 mg/ml hyaluronidase (Sigma Aldrich) for 20 min at 37 °C. Primary antibodies were detected using the appropriate Alexa Fluor-conjugated secondary antibody (Abcam). All sections were visualized using the ProLong Gold Antifade and examined under a fluorescence microscope (Zeiss).
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