Monolayer cell culture, Chondrocytes microspheres and pellets were fixed in 4% PFA (Santa Cruz) at room temperature in dark for 30 minutes. Cartilage tissues were fixed in 4% PFA (Santa Cruz) at room temperature in dark overnight. Except monolayer culture, other samples were cryo-embedded and cut into 20 µm thick frozen sections. The sections were first incubated overnight at 4 °C with primary antibodies against Sox9 (ab76997), collagen II (ab34712), Runx2 (ab76956) and Collagen X (ab49945) respectively, followed by incubation with an Alexa Fluor 488-conjugated secondary antibodies (Abcam) in dark for 30 minutes. Fluorogel with Dapi (EMS, USA) is used to mount cover glass on sections and counterstain the nucleus. Images were taken using an inverted confocal microscopy (LSM710, Carl Zeiss, Germany).
Alexa fluor 488 conjugated secondary antibody
Manufactured by Abcam
Sourced in United States, United Kingdom, Germany
Alexa Fluor 488-conjugated secondary antibody is a fluorescent-labeled antibody used for the detection and visualization of target proteins in various applications such as immunofluorescence, flow cytometry, and Western blotting. It binds to the primary antibody and emits green fluorescence upon excitation, allowing for the specific labeling and detection of the target protein.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Beyotime (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (4 mentions)
Triton x 100, by Merck Group (3 mentions)
Fluorescence microscope, by Olympus (3 mentions)
Accuri c6 plus, by BD (3 mentions)
Guava easycyte, by Merck Group (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Fv3000, by Olympus (2 mentions)
Slowfade gold antifade reagent, by Thermo Fisher Scientific (2 mentions)
Anti α sma, by Abcam (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Permeabilization wash buffer, by BioLegend (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Lsm 710, by Zeiss (1 mentions)
Paraformaldehyde (pfa), by Santa Cruz Biotechnology (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
67 protocols using alexa fluor 488 conjugated secondary antibody
1
Immunohistochemistry Analysis of Chondrocytes
2
Investigating Cellular Stress Responses
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Dulbecco’s modified Eagle medium (DMEM), foetal bovine serum (FBS) and antibiotics (penicillin and streptomycin) required for the cell cultures were obtained from GIBCO (Carlsbad, CA, USA). The cell culture plates were manufactured by Corning Inc. (Corning, NY, USA). The CCK-8 and DAPI working solution were purchased from Beyotime Biotechnology (Shanghai, China). l -glutamine, tunicamycin (TUNI), doxorubicin (DOX) and dimethylsulfoxide (DMSO) were purchased from Sigma (Saint Louis, MO, USA). The antibodies against XBP-1s and phospho-PERK were obtained from Santa Cruz Biotechnology, and the antibodies against PERK, ATF6, IRE1α, GRP78, CHOP/GADD153, ATF4 cleaved- caspase3, and β-actin, plus the Alexa Fluor 488-conjugated secondary antibodies, were obtained from Abcam (Cambridge, MA, USA).
3
Fluorescent Labeling of Cell Markers
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All DNA oligonucleotides, including three probes for rat lncRNA malat1 (Malat1-a, -b and -c) and one probe for GAPDH (positive control; Table I ), were designed and synthesized and labeled on the 5′-end with Alexa Fluor 633 by Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Probes were stored at −20°C in powder form. To prepare a 250 nM working solution, probes were diluted in hybridization buffer. Antibodies against NeuN, Iba1 and GFAP were used as biomarkers of neurons, microglia and astrocytes, respectively (11 (link)). The primary and Alexa Fluor 488-conjugated secondary antibodies used for immunofluorescence were purchased from Abcam (Cambridge, MA, USA; Table II ) and diluted with blocking buffer to 1:500 and 1:400, respectively.
4
Hippocampal Microglial Immunofluorescence Staining
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Immunofluorescence staining of the hippocampal slices was performed as described in a previous study by our group (15 (link)). All hippocampal sections were incubated with 3% bovine serum albumin (OriGene Technologies, Inc.) for 20 min at room temperature (25-30˚C) to block non-specific binding sites. The sections were then incubated with the primary antibody, rabbit polyclonal anti-ionized calcium-binding adaptor molecule-1 (Iba1; cat. no. ab153696; 1:500; Abcam), for 12 h at 4˚C. The sections were then incubated with Alexa Fluor 488-conjugated secondary antibodies (cat. no. ab150077; 1:100; Abcam) for 2 h at room temperature (25-30˚C), followed by counterstaining with DAPI. Representative fluorescence microscopy images of the hippocampal samples were taken and immunofluorescence staining intensity was quantified using a Nikon Eclipse Ni-U microscope (Nikon Corporation) at x10 magnification and image pro plus software version 6.0 (Media Cybernetics, Inc.) by an investigator who was blinded to the origin of the sections. Integrated optical density of each slice from each group was measured and presented as a percentage with CB2R WT + O2 as the control. Microglial morphology was analyzed using Imaris software (version 8.1; Oxford Instruments) at x100 magnification to assess the number of microglial branches.
5
Immunofluorescence Visualization of PTMA and HMGB1
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Following immobilization and permeabilization of the cells with 4% paraformaldehyde and 0.1% Triton X-100 for 15 min, respectively, the overnight cultivation of the cells impeded by 10% goat serum with PTMA (1:300) and HMGB1 (1:300) antibodies was carried out at 4 ℃, followed by the exposure to Alexa Fluor®594 and Alexa Fluor® 488-conjugated secondary antibodies (1:400; Abcam, Cambridge, UK) for 90 min. Finally, Hoechst (Beyotime) staining lasted for 5 min. The distribution of PTMA and HMGB1 were tracked using the Olympus fluorescence microscope.
6
Evaluating EMT Markers in A549 Cells
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To examine the effect of O-PMs on the in situ expression of EMT markers and fibronectin, confluent A549 cells on sterilized-coverslip in 12-well plate were incubated with 1 mL DMEM medium containing 10% FBS with or without adding 100 μg/mL of O-PMs for 24 h by immunocytochemistry. The cells were fixed in 4% paraformaldehyde, permeabilized with 0.01% Triton X-100, blocked with 1% bovine serum albumin (BSA) in PBS, and then incubated with the indicated primary antibodies at 4 °C overnight. After being washed with PBS, the cells were incubated with AlexaFluor 488 conjugated secondary antibodies (Abcam), and then observed and photographed with a fluorescence microscope. DAPI was used for nuclear counterstaining.
7
Immunofluorescent Labeling of Cochlear Hair Cells
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Cultured cochlear explants were fixed with 4% paraformaldehyde for 15 min and washed three times for 15 min each in phosphate-buffered saline (PBS). They were then blocked for 1 h in PBS containing 0.1% Triton X-100, 5% donkey serum, and 1% bovine serum albumin (BSA). The explants were incubated overnight at 4°C with anti-myosin-VIIa antibody (1:500; Proteus Biosciences, Ramona, CA, United States), followed by Alexa Fluor 488-conjugated secondary antibody (1:200; Abcam, Cambridge, MA, United States) and Alexa Fluor 546 phalloidin (1:1000; Thermo Fisher Scientific, Waltham, MA, United States) to label hair cells; they were then examined by confocal microscopy (LSM 880; Zeiss, Oberkochen, Germany). Photographs were taken from the same area of tissue in each group (middle turn of the cochlea). The number of hair cells that were immunopositive for myosin VIIa antibody and phalloidin were counted.
8
PTEN Protein Expression Quantification
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Cells were permeabilized using 0.1% Triton X-100 for 5 min on ice. Then cells were incubated with PTEN antibody (Abcam, Cambridge, MA, USA) at 1: 120 for 30 min at room temperature, and Alexa Fluor 488-conjugated secondary antibody (Abcam) was used at 1: 2000 at room temperature for 30 min. Finally, the cells were maintained at 4°C and the PTEN−/+ fraction was sorted using a FACSAria flow cytometer (BD Biosciences, San Jose, CA, USA). Data analysis was performed using FlowJo version 4.
9
Immunofluorescence Staining of Pluripotent Stem Cells
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ES cells were plated on gelatinized glass coverslips on primary mouse embryonic fibroblasts. Cells were fixed with 4% PFA for 30 min followed by permeabilizing with 0.5% Triton X-100/PBS for 20 min at RT. Cells were blocked in blocking buffer (3% BSA, 2% donkey serum in PBS) for 10 min and stained with a primary antibody for 1 h at 37 °C. After washing three times for 10 min with PBS, cells were stained with a secondary antibody for 1 h at 37 °C. Antibodies used: For LIN28A (rabbit, Cell Signaling), a rabbit antibody at 1:300 and a donkey anti-rabbit Alexa Fluor 488-conjugated secondary antibody (abcam) at 1:400 were used. For Fibrillarin (mouse, abcam) and NPM1 (mouse, Sigma-Aldrich), the mouse antibodies at 1:200 and a DyLight 594 goat anti-mouse secondary antibody (EarthOx) at 1:400 were used. Following washing for another three times with PBS, DAPI was used for nucleus staining. Cells were then imaged using Zeiss LSM880 fluorescence microscope at a 63× oil objective. For high regulation microscopy imaging, LSM800 with Airyscan module was used.
10
Quantifying γ-H2AX Protein Expression
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γ-H2AX levels were determined as previously described [17 (link)]. Briefly, target cells were fixed in 70% ethanol over a 4-h period at 4˚C, following fixing in formaldehyde solution on ice. Cells were washed with PBS buffer and 1% BSA solution and then immersed in 100 µl 1% BSA reagent. The primary antibody (1:100; Abcam) against γ-H2AX (phospho S139) protein and the Alexa Fluor® 488-conjugated secondary antibody (1:100; Abcam) were used for the detection of γ-H2AX protein expression. The fluorescence in the dye was excited and detected on a BD Accuri C6 Plus System (BD Biosciences).
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