Brain tissues were cut into 35 µm thick slices until the entire structure of the hippocampus was detected. The slices were washed three times with 0.1 M phosphate-buffered saline (PBS) and incubated at room temperature for 20 minutes in 5% bovine serum albumin. Primary (overnight, 4°C) and secondary antibody incubations (one hour, room temperature) were carried out. The slices were washed three times with PBS after each incubation. Primary antibody was mouse anti-NR2B 1 : 200 (ab93610, Abcam). Secondary antibody was goat anti-mouse immunoglobulin G (Alexa Fluor 488) 1 : 2000 (ab150157, Abcam). Slices were coverslipped with a water-based mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) (KeyGEN BioTECH, Jiangsu, China). Stained sections were then observed by fluorescence microscopy (Olympus) and processed with ImageJ Software.
Ab150157
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab150157 is a laboratory equipment product. It is designed for use in scientific research and experiments. The core function of this product is to perform a specific task or measurement required in a laboratory setting. No further details about the intended use or interpretation of this product are provided.
Automatically generated - may contain errors
Lab products found in correlation
Ab6326, by Abcam (5 mentions)
Ab150079, by Abcam (5 mentions)
Ab150075, by Abcam (4 mentions)
Lsm 510 meta confocal microscope, by Zeiss (4 mentions)
Ab150116, by Abcam (3 mentions)
Ab150080, by Abcam (3 mentions)
Ab6640, by Abcam (3 mentions)
Ab150077, by Abcam (3 mentions)
Alexa fluor 488, by Abcam (3 mentions)
Ab150078, by Abcam (2 mentions)
Ab150115, by Abcam (2 mentions)
Ab104139, by Abcam (2 mentions)
Ab150083, by Abcam (2 mentions)
Ab64693, by Abcam (2 mentions)
Ab150073, by Abcam (2 mentions)
Fluorescence microscope, by Nikon (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Ab93610, by Abcam (1 mentions)
36 protocols using ab150157
1
Hippocampal NR2B Subunit Localization
2
Quantifying Cell Proliferation using BrdU Labeling
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For BrdU (5-bromo-2′-deoxyuridine) labelling, mice were intraperitoneally injected with 10 μl per gram body weight of BrdU labeling reagent (#000103, Invitrogen) 6 h before being sacrificed. The hindlimbs were then harvested and processed for paraffin sections. To detect BrdU-labeled proliferating cells, tibial sections were dewaxed, rehydrated, and then denatured with 2 N HCl, prior to antigen retrieval with 0.125% trypsin in PBS at 37°C for 10 min. After blocking with 10% goat serum for 1 h, sections were incubated with rat anti-BrdU antibody (ab6326, Abcam, 1:100) at 4°C overnight, followed by incubation with Alexa Fluor 647-conjugated donkey anti-rat (ab150155, Abcam,1:200) or Alexa Fluor 488-conjugated goat anti-rat secondary antibody (ab150157, Abcam, 1:200). Finally, nuclei were counterstained with DAPI (C1005, Beyotime Biotechnology, Shanghai, China) to stain the cell nuclei, and subsequently mounted with anti-fade mounting medium.
3
DNA Replication Dynamics Analysis
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Exponentially growing HCT116 cells were labeled with the medium containing 25 μM thymidine analog iododeoxyuridine (IdU; CSN12762, CSNpharm) for 30 min. Cells were then washed with PBS before the addition of warm media containing 250 μM chlorodeoxyuridine (CldU; C6891, Sigma) for another 30 min. Labeled cells were washed with PBS twice, harvested, and lysed on slides, and DNA was stretched by tilting the slides. The DNA was fixed in the 3:1 methanol/acetic acid solution and then airdried completely. Next, fibers were denatured using 2.5 M HCl for 80 min and blocked with 5% BSA. To detect the incorporation of CldU and IdU in DNA fibers, coverslips were incubated with rat anti-BrdU (ab6326, Abcam) and mouse anti-BrdU (B2531, Sigma) primary antibodies for 2 h. The slides were stained with goat anti-rat Alexa Fluor 488 (ab150157, Abcam) and goat anti-mouse Alexa Fluor 594 (ab150116, Abcam) secondary antibodies for 1 h. Slides were mounted with an anti-fade solution and images were acquired using a confocal microscope. Fiber lengths were analyzed using ImageJ.
4
Multimodal Characterization of Vascular Development
5
Immunofluorescent Analysis of Macrophage Response in Experimental Periodontitis
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The maxillae were dissected at 0, 3, 6, and 10 days after experimental periodontitis treatment and were subsequently fixed with 4% paraformaldehyde. For tissue immunofluorescent staining, the maxillae were then decalcified with 0.5 M ethylenediaminetetraacetic acid (EDTA) for 48 h and embedded at −80°C with compound (OCT, Sakura Finetek, Torrance, CA, USA). Frozen sections were sliced at −25°C and stored at −20°C. After antigen repairing and blocking for 1 h at room temperature in 3% bovine serum albumin (BSA), the slices were incubated overnight using the primary antibody of rat-anti-mouse anti-F4/80 (Abcam Cat# ab16911, RRID:AB_443548 ). Then, goat-anti-rat Alexa Fluor® 488 secondary antibodies (Abcam Cat#ab150157, RRID:AB_2722511 ) were used at 1/200 dilution to further stain the slices on the following day. The slices were counterstained with Hoechst 33342 Staining Dye Solution (ab228551) for 10 min at 25°C. Laser-scanning confocal microscopy was employed to detect the fluorescent distribution.
6
Klotho and CCL5 Expression Analysis
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Dewaxed kidney tissue sections were rehydrated, heated for 20 min in a microwave for antigen retrieval, and then incubated with immunohistochemical serum blocking agent (GEPbio, 317615) for 30 min at 37°C to block non-specific staining. Subsequently, the sections were incubated with a rabbit anti-Klotho antibody (ab181373, Abcam) and rat anti-CCL5 antibody (NB120-10394, NOVUS) overnight at 4°C. The next day, the sections were washed with PBS for three times and then incubated with an Alexa Fluor 555-labeled anti-rabbit second antibody (ab150078, Abcam) and Alexa Fluor 488-labeled anti-rat second antibody (ab150157, Abcam) at 37°C for 30 min. Then, cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, GEPbio, 721621) for 3 min. Finally, the sections were washed with PBS again and mounted with anti-fade medium (GEPbio, 717615). Stained sections were examined and imaged under a fluorescence microscope. Mean fluorescence intensity (MFI) of each section was analyzed by Image J software.
7
Histological Analysis of Skeletal Muscle
8
Immunohistochemical Analysis of ETV6 and F4/80 in Aorta
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Aortas were fixed in 4% formaldehyde for 24 h, dehydrated overnight, and embedded in paraffin. Paraffin cross-sections (4-μm thick) were prepared. After incubation with 5% normal goat serum for 1 h at room temperature, the sections were incubated with rabbit anti-ETV6 antibody (1:200, PA5-109,697, Thermo Fisher Scientific) and rat anti-F4/80 (10 μg/ml, ab6640, Abcam) overnight at 4oC. After three washes with PBS, the samples were incubated with 5 μg/ml Alexa Fluor® 488-conjugated goat anti-rat IgG H&L and Alexa Fluor® 594-conjugated goat anti-rabbit IgG H&L (1:1000, ab150157, ab150080, Abcam) for an hour at room temperature. For Immunofluorescence cytochemistry, macrophages were fixed in acetone for 10 min and incubation with 5% normal goat serum for 1 h at room temperature. Macrophages were then incubated with NF-κB p65 antibody (1:500, #8242, Cell Signaling Technology) overnight at 4oC. After three washes with PBS, cells were incubated with Alexa Fluor® 488-conjugated goat anti-rabbit IgG H&L (1:1000, ab150077, Abcam) for an hour at room temperature. Cells were washed and incubated in the mounting medium with DAPI (ab104139, Abcam). The samples were observed and photographed on a Leica DM500 fluorescence microscope (Leica).
9
Cathepsin B Regulates Adipogenesis
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3T3L1/Mock, 3T3L1/CTSB-overexpressing (OE), and 3T3L1/CTSB-mCherry-OE pre-adipocytes grown on cover slips were differentiated into adipocytes and treated with or without 10 μM of the cathepsin B inhibitor CA074ME (4323-v, Peptide Institute) for 24 h. After fixing with 4% paraformaldehyde for 15 min, cells were permeabilized and incubated with primary antibodies against PLIN1 (ab61682, 1:400, Abcam) or LAMP2 (ab13524, 1:1000, Abcam) at 4 °C overnight, and then with Alexa Fluor-conjugated secondary antibodies (Alexa Fluor-488 goat anti-rabbit IgG (H + L), A11070, 1:1000, Invitrogen, Carlsbad, CA, USA; Alexa Fluor-594 goat anti-rabbit IgG (H + L), A110121, Invitrogen; Alexa Fluor-488 goat anti-rat IgG (H + L), ab150157, Abcam) for 1 h at room temperature. Hoechst 33342 (H3570, 1:10000, Invitrogen) and Nile red were used to stain nuclei and LDs, respectively. Images were acquired on an SP8 confocal microscope (Leica, Wetzlar, Germany) at 63 × magnification.
10
Immunofluorescence Staining of α-SMA and Desmin
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Immunofluorescence staining assays were performed as we described previously [15 (link)]. Cells were incubated overnight with the primary antibody against α-SMA (ab5694) and desmin (ab15200) (1 : 50 dilution; Abcam, USA) at 4°C. The cells were washed three times with PBS (5 min each) and then incubated in the dark for 1 h at room temperature with Alexa Fluor 488 and 550 conjugated goat anti-rat secondary antibody (1 : 200 dilution; ab150157 and ab150083, Abcam, USA). Nuclei of cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, USA). Images were obtained using a Zeiss LSM 510 META Confocal microscope using 20x/0.5 w and 40x/1.2 w objectives.
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