Cochlear sections and spreads were incubated with 5% normal goat serum (ZSGB-BIO, Beijing, China) and 0.3% Triton X-100 (Sigma-Aldrich, St. Louis, MO) in phosphate-buffered saline (PBS) for 2 h at room temperature. After washing three times with PBS, the samples were incubated with primary antibody solution at 4°C overnight. After multiple washes, the samples were incubated with secondary antibodies at a ratio of 1:300 for 2 h at room temperature and protected from light. The primary antibodies used were anti-myosin-VIIa (1:300, Proteus BioSciences Inc. Ramona, CA), anti-CtBP2 (1:500, BD Biosciences, Franklin Lakes, NJ), anti-GluR2 (1:400, Millipore, Burlington, MA), anti-8-hydroxy-2′-deoxyguanosine (8-OHdG, 1:300, Abcam, Cambridge, United Kingdom), anti-green fluorescent protein (GFP) (1:100, Santa Cruz Biotechnology, Dallas, TX), and anti-4-HNE (1:500; Abcam). The secondary antibodies used were goat anti-mouse IgG1 Alexa Fluor 568, goat anti-mouse IgG2a Alexa Fluor 488, and goat anti-rabbit IgG (H + L) Alexa Fluor 647 (1:300, Invitrogen/Molecular Probes, Eugene, OR). 4, 6-diamidino-2-phe-nylindole (DAPI) was used for the final addition of coverslips. The expression of 8-OHdG and 4HNE was analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc. United States).
Alexa fluor 488 goat anti mouse igg2a
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 488 goat anti-mouse IgG2a is a fluorophore-conjugated secondary antibody used for detection and visualization of mouse IgG2a primary antibodies in various immunoassay applications. It exhibits green fluorescence upon excitation.
Automatically generated - may contain errors
Lab products found in correlation
B 2 mouse monoclonal, by Santa Cruz Biotechnology (4 mentions)
Alexa fluor 647 goat anti mouse igg1, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 488 goat anti mouse igg1, by Thermo Fisher Scientific (3 mentions)
Detachkit, by Thermo Fisher Scientific (2 mentions)
Lsrfortessa flow cytometer, by BD (2 mentions)
Efluor 450, by Thermo Fisher Scientific (2 mentions)
40 μm cell strainer, by Corning (2 mentions)
Paraformaldehyde (pfa), by Merck Group (2 mentions)
Tryple, by Thermo Fisher Scientific (2 mentions)
Fix perm kit, by Thermo Fisher Scientific (2 mentions)
Goat anti mouse igg2b alexa fluor 647, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Ab56416, by Abcam (2 mentions)
Facscanto 2, by BD (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Alexa fluor 647 donkey anti goat igg, by Thermo Fisher Scientific (2 mentions)
Mouse igg1 anti ctbp2, by BD (2 mentions)
Alexa fluor 555 goat anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 555 goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
25 protocols using alexa fluor 488 goat anti mouse igg2a
1
Immunohistochemical Analysis of Cochlear Tissues
2
Immunostaining of Myeloid Subsets in Tumors
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Characterization of TIM was carried out with triple immunofluorescent staining as described previously.24 (link) Briefly, after deparaffinization and rehydration of the 4-μm tissue sections, heat-mediated antigen retrieval with a 1 mmol/L EDTA solution (pH 9.0) was performed. A mixture containing primary antibodies anti-CD33 (1:50, mouse-IgG2b, clone PWS44, Leica Microsystems B.V.), anti-CD14 (1:100, mouse-IgG2a, clone 7, Leica Microsystems B.V.) and anti-CD163 (1:400, mouse-IgG1, Clone 10D6, Leica Microsystems B.V.) was applied to the tissue sections overnight at room temperature. The next day, a mixture of fluorescently labeled secondary antibodies (goat anti-mouse IgG2b-Alexa Fluor 546, goat anti-mouse IgG2a-Alexa Fluor 488 and goat anti-mouse IgG1-Alexa Fluor 647; Molecular Probes) was used to detect primary antibody binding. Images were captured with a confocal laser scanning microscope (Zeiss LSM 510) in a multitrack setting. Epithelial tumor cell nests and stromal areas were measured using the Zeiss LSM Image Examiner. Myeloid subsets were manually counted in all representative images for either tumor epithelium, stroma, or both and were presented as the number of cells per mm2.
3
Evaluation of LAC-CM-EVs Uptake
4
Evaluation of LAC-CM-EVs Uptake
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For LAC-CM-EVs uptake evaluation, the cells were incubated with 1010 particles per mL of LAC-CM-EVs overnight. The next day, the CMs were dissociated into single cells with TrypLE (Thermo Fisher Scientific), while hCMEC and hCFib were dissociated using PromoCell DetachKit and 0.25% trypsin, respectively. Subsequently, the cells were incubated with a fixable viability dye eFluor 450 (eBiosciences), washed in D-PBS, filtered using 40 μm cell strainer (Corning) and fixated using 4% (v/v) paraformaldehyde (Sigma-Aldrich) in D-PBS. For analysing intracellular cardiac troponin T (cTnT) expression, the CMs were further permeabilized using the FIX & PERM kit (Thermo Fisher Scientific) and stained with a primary antibody mouse anti-human cardiac troponin T (1 : 100; R&D systems), followed by a secondary antibody goat anti-mouse IgG2a Alexa Fluor 488 (1 : 200; Thermo Fisher Scientific) at 4 °C. Analytical flow cytometry was performed using LSR Fortessa flow cytometer (BD Biosciences), and analysis was carried out with FlowJo V10 software.
5
Immunofluorescent Labeling of Dopaminergic and Alpha-Synuclein Proteins
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Cells were fixed with 4% PFA and blocked with 2% donkey serum (Sigma) in PBS‐T (0.1% Triton X‐100 (Fisher) in PBS (Thermo Fisher Scientific)). Primary antibodies used include tyrosine hydroxylase (1:1,000, rabbit, Millipore), FOXA2 (1:100, goat, Santa Cruz), α‐synuclein (1:250, mouse IgG2a, Abcam), α‐synuclein (1:250, mouse IgG1, BD), phosphoserine‐129 α‐synuclein (pS129‐αSyn, 1:1,000, rabbit, Abcam) and β‐III tubulin (1:1,000, mouse IgG2b, Abcam). Secondary antibodies including donkey anti‐rabbit IgG Alexa Fluor‐488 (Thermo Fisher Scientific), donkey anti‐goat IgG Alexa Fluor‐568 (Thermo Fisher Scientific), donkey anti‐mouse IgG Alexa Fluor‐647 (Abcam), goat anti‐mouse IgG2a Alexa Fluor‐488 (Thermo Fisher Scientific), goat anti‐mouse IgG1 Alexa Fluor‐488 (Thermo Fisher Scientific), goat anti‐mouse IgG2b Alexa Fluor‐647 (Thermo Fisher Scientific) were used at 1:1,000, while DAPI (Thermo Fisher Scientific) was used at 1:10,000. Images were acquired on the Axio Observer (Zeiss) or the Eclipse Ti‐E (Nikon) microscope.
6
Antibodies for Immunoblotting and Microscopy
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Antibodies employed for immunoblotting and microscopy are as follows: rabbit anti-Myc (Bethyl), rabbit anti-TOMM20 (FL-145), mouse anti-β-actin (C-4), mouse anti-MAVS (E3), mouse anti-Mfn2 (XX-1), mouse anti-calnexin (E-10), mouse anti-Tim23 (H-8) (all from Santa Cruz, CA, USA), mouse anti-FLAG (M2) (Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-CARD19 (HPA010990), rabbit anti-SAMM50 (HPA034537) (Atlas Antibodies, Stockholm, Sweden), mouse anti-mitofilin (2E4AD5), mouse anti-ATP synthase β (4.3E8.D10) (both from Thermo Fisher, Waltham, MA, USA), and rat anti-OLLAS ([32 (link)]; purified from hybridoma supernatant). Secondary antibodies for microscopy were goat anti-rabbit (H + L) Alexa Fluor 647, goat anti-mouse (IgG1) Alexa Fluor 488 and goat anti-mouse (IgG2a) Alexa Fluor 488 (all from Thermo Fisher, Waltham, MA, USA), used at a 1:200 dilution.
7
Immunofluorescent Staining of Human CNS Tissue
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Human CNS tissue was fixed with 4% paraformaldehyde (PFA) and embedded in paraffin as described previously (77 (link)), and 2-μm-thin sections were cut. Tissue sections were deparaffinized, and antigen retrieval [Marmite Pascal tris-EDTA (pH 9.0), 125°C, 30 s] was performed. To prevent unspecific binding of primary antibody, tissues were incubated with normal goat serum (10% in PBS) before overnight incubation with a mouse immunoglobulin G2a (IgG2a) anti-H3K9me2 (1:200; Abcam, ab1220) and a mouse IgG2b anti-HuC/D (1:100; Invitrogen, A-21271) antibody. Autofluorescence was removed (Merck, catalog no. 2160), and primary antibodies were visualized using goat anti-mouse IgG2a Alexa Fluor 488 (Invitrogen, A21131) and goat anti-mouse IgG2b Alexa Fluor 647 (Life, A21242) (see table S3 for antibodies). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen, D3571). All steps were performed at room temperature. Stained sections were scanned using the Pannoramic 250 FLASH II (3DHISTECH) Whole Slide Scanner at a resolution of 0.221 μm per pixel. Their use for scientific purposes was in accordance with institutional ethical guidelines and approved by the ethics committee of the University of Geneva (Switzerland). Informed consent was obtained from subjects, in accordance with approval of the local ethical committee.
8
Feline Immune Cell Phenotyping
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Monoclonal antibodies against the epsilon chain of feline CD3 (NZM1) and against feline CD56 (SZK1) were kindly provided by Dr. Yorihiro Nishimura (Tokyo University, Japan)45 (link). Monoclonal antibodies FE5.4D2, and CA2.1D6 recognising feline CD8β, and canine CD21, respectively, were purchased from Bio-Rad. A monoclonal antibody (FJK-16s), directly conjugated with Alexa fluor 647 (AF647) and cross-reacting with feline Foxp3 was purchased from eBioscience. Monoclonal antibody CAT30A against feline CD4 was purchased from Veterinary Medical Research and Development (VMRD). Conjugated secondary antibodies (Invitrogen) were goat anti-rat Alexa Fluor 488, goat anti-mouse IgG R-Phycoerythrin, goat anti-mouse IgG2a Alexa Fluor 488, goat anti-mouse IgG1 Alexa Fluor 647 and goat anti-mouse IgG3 fluorescein isothiocyanate (FITC). When primary antibodies from the same IgG1 isotype were used, one primary antibody was labeled with Zenon Alexa Fluor 488 Mouse IgG1.
9
Quantifying Intracellular p62 Accumulation
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Intracellular p62 accumulation was measured in Caco-2 cells treated with BBM (36.7 μM), or DAS (30 μM), or left untreated, for 72 h. Cells fixed in 4% paraformaldehyde (PFA; Electron Microscopy Sciences) were permeabilized with PBS containing 0.5% saponin and 1% bovine serum albumin (BSA; both Sigma), followed by staining with either mouse anti-p62 (IgG2a, 5 μg/mL, Abcam ab56416) or mouse IgG2a isotype control (5 μg/mL, eBioscience 14-4724-85), and thereafter staining with goat anti-mouse IgG2a-Alexa Fluor 488 (Invitrogen A11029). Intracellular p62 accumulation was quantified using flow cytometry (FACSCanto II, BD Biosciences).
10
Quantification of Intracellular p62 Levels
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Cells were fixed in 4% paraformaldehyde (PFA; Electron Microscopy Sciences) and subsequently permeabilized with PBS containing 0.5% saponin and 1% bovine serum albumin (BSA; both Sigma). Permeabilization was followed by staining with either mouse anti-p62 (IgG2a, 5 mg/mL, Abcam ab56416) or mouse IgG2a isotype control (5 mg/mL, eBioscience 14-4724-85), and thereafter staining with goat anti-mouse IgG2a-Alexa Fluor 488 (Invitrogen A11029). Intracellular p62 levels were quantified using flow cytometry (FACSCanto II, BD Biosciences).
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