The 293T cells were cultured at coverslips and transfected with RPL15-mCherry_4 and RPL-histone3.3 mRNA by TransIT, and fixed by 4% PFA for antibody immunofluorescence staining after 24 h of transfection. The samples were incubated in primary antibodies (1:500) for 3 h and secondary antibodies conjugated with Alexa Fluor 647 (1:500) for 2 h, with 15 min DAPI staining before being mounted in Prolong gold antifade reagent (ThermoFisher Scientific, P36934). For SiRNeoblasts, 20,000 cells were sorted out for each group and transfected with RPL15-mCherry_4 and RPL-histone3.3 mRNA by TransIT. At 24 hpt, following fixed by 4% FA in 0.4X PBS twice for 10 min, incubated in Hybe at 56°C for 2 h and blocked by 10% Horse serum in PBSTx0.1% at room temperature for 30 min, the cells were stained with primary antibodies (1:500) and secondary antibodies conjugated with Alexa Fluor 555 (1:500) for 2 h, respectively. The antibodies included rabbit polyclonal mCherry antibody (MBL, PM005), mouse monoclonal Flag antibody clone M2 (Sigma, F1804), goat anti-mouse IgG antibody (H + L) Alexa Fluor 647 (Abcam, ab150119), goat anti-rabbit IgG antibody (H + L) Alexa Fluor 647 (Abcam, ab150083), goat anti-mouse IgG antibody (H + L) Alexa Fluor 555 (Abcam, ab150118) and goat anti-rabbit IgG antibody (H + L) Alexa Fluor 555 (Abcam, ab150086).
Ab150119
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab150119 is a rabbit monoclonal antibody directed against the human TrkA (NTRK1) protein. TrkA is a receptor tyrosine kinase that binds to nerve growth factor and is involved in neuronal growth and survival. This antibody can be used for various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab150081, by Abcam (4 mentions)
Ab150086, by Abcam (2 mentions)
Ab150083, by Abcam (2 mentions)
Ab150118, by Abcam (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Ab6721, by Abcam (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (2 mentions)
Ab150077, by Abcam (2 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Lsm 880, by Zeiss (1 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (1 mentions)
Ab228549, by Abcam (1 mentions)
Dm6b microscope, by Leica (1 mentions)
Ab7817, by Abcam (1 mentions)
Ab28364, by Abcam (1 mentions)
Ab92486, by Abcam (1 mentions)
F6140, by Merck Group (1 mentions)
S 1000, by Vector Laboratories (1 mentions)
Lsm 780 fcs confocal microscope, by Zeiss (1 mentions)
H 1500, by Vector Laboratories (1 mentions)
15 protocols using ab150119
1
Protein Localization in Transfected Cells
2
Immunofluorescent Analysis of Murine Inner Ear
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Neonatal mice were sacrificed by decapitation, and adult mice were sacrificed by CO2 inhalation. Inner ears were dissected under operating binoculars and fixed in 4% PFA for 2 h at room temp. After fixation, inner ears were washed in PBS and stored in 4°C until dissection. Inner ears of mice older than P10 were decalcified in 0.25 M EDTA until entirely soft. The organ of Corti was dissected in PBS under operating binoculars. Specimens were permeabilized and blocked in 2% Triton X‐100 and 10% normal goat serum for 2 h at room temperature, and were then incubated overnight at 4°C in the appropriate primary antibody diluted in Phosphate Green antibody diluent (Bar Naor Ltd) according to manufacturer instructions. Specimens were washed and incubated for 2 h at room temperature in the relevant secondary antibody diluted in PBS according to manufacturer instructions and then mounted in ProLong Gold Antifade Mountant (Thermo Fisher Scientific) and imaged using a Zeiss LSM 880 (Zeiss, Oberkochen, Germany) equipped with an Airyscan detector. Antibody and stain concentrations were as follows: rabbit polyclonal myosin VIIa (Proteus Biosciences 25‐6790) 1:250, mouse anti‐FLAG (Sigma F3165) 1:1,000, DAPI (Abcam ab228549) 1:1,000, goat anti‐mouse (Abcam ab150119) 1:250, and goat anti‐rabbit Alexa Fluor 488 (Cell Signaling 4412s).
3
Immunohistochemical Analysis of Murine Osteoarthritis
4
Quantifying Cellular Proliferation via Ki-67
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Cellular proliferation was assessed using Ki-67 staining. Human RCFs (1 × 105) were seeded in a 35mm confocal dish. After 24 h incubation, the study groups were exposed to various studied agents, according to each study’s subset. A fixative solution of cold methanol was added to each well, which was then incubated for 20 min at 4 °C. The cells were permeabilized with 0.3% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) added to each well, which was then incubated for 20 min at room temperature. The cells were then incubated in 1% bovine serum albumin (Amresco, Solon, OH, USA) in PBS for 1 h at room temperature. After that, the 1:200 diluted Ki-67 (ab15580, Abcam, Cambridge, MA, USA) and β-actin (Thermo Fisher Scientific, Waltham, MA, USA) primary antibody was added, and the cells were incubated for 2 h at room temperature. The secondary antibodies (ab150119, Goat Anti-Mouse IgG H&L Alexa Fluor® 647/ab150081, Goat-Anti-Rabbit IgG H&L Alexa Fluor® 488, abcam, Cambridge, MA, USA) were used at the 1:200 dilutions for 1 h at room temperature and cells were counterstained with 1 μg/mL of DAPI (4′,6-diamidino-2-phenylindole, Thermo Fisher Scientific, Waltham, MA, USA). The cells were then evaluated through a fluorescence microscope (Nikon, Ti2-U FL, Tokyo, Japan); digital photographs were taken at 200 magnifications.
5
Osteoporosis Treatment Protocol in Rats
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Twenty-four female Wistar rats were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. (Shandong, China). All animal experiments were approved by the Institutional Animal Care and Use Committee, School and Hospital of Stomatology, Shandong University (No. 20210912).
ED-71 was purchased from Chugai Pharmaceutical Co., Ltd. According to the manufacturer’s instructions, it was dissolved in absolute ethanol for storage. Before use, the solution was diluted to the corresponding concentration. The Anti-Osterix antibody (ab209484), Anti-P16 antibody (ab54210) and secondary antibodies (ab6721, ab102448, ab150081, and ab150119) were purchased from Abcam (Cambridge, United Kingdom). The Anti-Runx2 antibody (20700-1-AP), Anti-P53 antibody (60283-2-Ig), Anti-GAPDH antibody (10494-1-AP), Anti-SIRT1 antibody (60303-1-Ig) and Anti-Nrf2 antibody (16396-1-AP) were purchased from Proteintech (Chicago, United States). The Anti-β-gal antibody (bs-4631R) and Anti-Osteocalcin (OCN) antibody (bs-4917R) was purchased from Bioss (Beijing, China). The Anti-P16 antibody (A0262) was purchased from ABclonal (Wuhan, China). EX-527 (HY-15452) and ML-385 (HY-100523) were purchased from MedChemExpress (New Jersey, United States).
ED-71 was purchased from Chugai Pharmaceutical Co., Ltd. According to the manufacturer’s instructions, it was dissolved in absolute ethanol for storage. Before use, the solution was diluted to the corresponding concentration. The Anti-Osterix antibody (ab209484), Anti-P16 antibody (ab54210) and secondary antibodies (ab6721, ab102448, ab150081, and ab150119) were purchased from Abcam (Cambridge, United Kingdom). The Anti-Runx2 antibody (20700-1-AP), Anti-P53 antibody (60283-2-Ig), Anti-GAPDH antibody (10494-1-AP), Anti-SIRT1 antibody (60303-1-Ig) and Anti-Nrf2 antibody (16396-1-AP) were purchased from Proteintech (Chicago, United States). The Anti-β-gal antibody (bs-4631R) and Anti-Osteocalcin (OCN) antibody (bs-4917R) was purchased from Bioss (Beijing, China). The Anti-P16 antibody (A0262) was purchased from ABclonal (Wuhan, China). EX-527 (HY-15452) and ML-385 (HY-100523) were purchased from MedChemExpress (New Jersey, United States).
6
Immunohistochemical Analysis of Neural Markers
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Six weeks after transplantation, the rats were sacrificed by injection of sodium pentobarbital, and the brain tissues were fixed with 4% paraformaldehyde. After being immersed in 15% and 30% sucrose in PBS, the brain tissues were sliced with a freezing microtome (20 μm thick). Cultured cells were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature. The fixed cells and brain sections were then permeabilized with 0.5% Triton X‐100 for 15 min at 37°C, blocked in 10% goat serum for 30 min at 37°C, and incubated overnight at 4°C with primary antibodies. They were then incubated with secondary antibodies in the dark at room temperature for 1 h. Then, the stained samples were counterstained with DAPI (P0131, Beyotime Biotechnology) and observed with a laser scanning confocal microscope. Images were taken with ZEN 2 software. The following antibodies were used: Nestin (1:50, abs137231, Absin, China), NSE (1:50, BA0535, Boster), GFAP (1:50, BA0056, Boster), CCR5 (1:50, sc‐17,833, Santa Cruz Biotechnology), TH (1:500, ab112, Abcam), CCL5 (1:50, sc‐365,826, Santa Cruz Biotechnology), goat anti‐rabbit IgG H&L (Alexa Fluor® 488,1:1000, ab150077, Abcam), goat anti‐mouse IgG H&L (Alexa Fluor® 647,1:1000, ab150119, Abcam), and goat anti‐rabbit IgG H&L (Alexa Fluor® 647,1:1000, ab150079, Abcam).
7
Immunofluorescent Imaging of ACE2, DC-SIGN, and SARS-CoV-2 Spike
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Cells were fixed and blocked with 5% BSA, and then incubated overnight at 4°C with rabbit anti-human ACE2 monoclonal antibody (ab108209; Abcam, UK), mouse anti-DC-SIGN monoclonal antibody (MAB161, R&D Systems, USA), mouse anti-L-SIGN monoclonal antibody (MAB162, R&D Systems, USA), or rabbit anti-SARS-CoV-2 spike monoclonal antibody (40150-R007, Sino Biological, China), depending on the experimental design. The cells were then incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG (ab150113; Abcam, UK), 488-conjugated goat anti-rabbit IgG (ab150081; Abcam, UK), Alexa Fluor 647-conjugated goat anti-mouse IgG (ab150119; Abcam, UK) or Alexa Fluor 647-conjugated goat anti-rabbit IgG (ab150075; Abcam, UK) for 1 h at room temperature. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI, Beyotime, China). Images were obtained with a Leica TCS SP8 laser confocal microscope (Leica Microsystems, Germany).
8
Immunofluorescence Staining of Cell Cultures
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The samples were washed twice with cold PBS after removing the culture medium. After that, the cells were fixed using 4% PFA in PBS for 20 min at room temperature and then washed three times with PBS. To permeabilize the samples, 0.1% Triton-X100 in PBS was added for 5 min, followed by three more washes with PBS. Next, the samples were treated with a blocking solution (2% BSA in PBS) for 1 h at room temperature. Primary antibodies were then diluted in a blocking solution according to the specifications shown in Table 1 , and then they were added to the samples for 1 h at room temperature or at 4 °C overnight. The following secondary antibodies were incubated for 1.5 h: goat anti-rabbit Alexa 488 (Abcam, ab150077, 1:250), goat anti-mouse Alexa 647 (Abcam, ab150119, 1:250), and goat anti-mouse Alexa 555 (Abcam, ab150118). Phalloidin staining was performed by adding phalloidin conjugated to iFluor 647 (Abcam, ab176759, 1:1000) or phalloidin conjugated to iFluor 555 (Abcam, ab176756, 1:1000). For nuclei detection, the samples were incubated for 5 min with Hoechst 33258 (1:20; Sigma). Images were taken using confocal microscopy (Nikon Eclipse Ni, Tokyo, Japan) or an inverted fluorescence microscope (Nikon Eclipse TI).
9
Immunofluorescence Analysis of ER Markers
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The primary antibodies used were β‐actin [anti‐β‐actin mouse mAb (8H10D10, #3700; Cell Signaling Technology, Danvers, Massachusetts)], anti‐BiP (rabbit Ab, 3177; Cell Signaling Technology), anti‐P4hb (rabbit Ab, ab137110; Abcam, Cambridge, UK), anti‐Calr (rabbit Ab, 2891; Cell Signaling Technology), anti‐GRP94 (rabbit Ab, 2104S; Cell Signaling Technology), anti‐GM130 (mouse Ab, 610 822; BD Biosciences, Franklin Lakes, New Jersey) and KDEL mouse antibody (2D6, MA5‐27581; Invitrogen, Carlsbad, California). The secondary antibodies used were goat anti‐rabbit IgG H&L Alexa Fluor 488 conjugated (ab150081; Abcam), goat anti‐mouse IgG H&L Alexa Fluor 647‐conjugated (ab150119; Abcam), HRP‐conjugated rabbit anti‐mouse IgG H&L (ab6728; Abcam) and goat anti‐rabbit IgG H&L (HRP) (ab6721; Abcam). The ER was stained using the ER‐ID Red assay kit, in accordance with the protocol of the manufacturer (Enzo Life Sciences, Farmingdale, New York).
10
Immunofluorescent Phenotyping of Macrophages
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Cells were fixed with 4% paraformaldehyde for 20 min. Then, they were blocked with 5% BSA in PBS for 1 h and incubated with the following specific antibodies at 37 °C for 1 h. The primary antibodies were rabbit anti-CD163 (1:300; ab182422, Abcam, UK) and mouse anti-CD206 (1:300; 60143-1-Ig, Proteintech, USA). Subsequently, cells were then incubated with goat anti-rabbit IgG H&L (Alexa Fluor® 488) (1:200; ab150081, Abcam, USA) and goat anti-mouse IgG H&L (Alexa Fluor® 647) (1:200; ab150119, Abcam, USA) for 1 h. Nuclei were stained with DAPI, and images were obtained by fluorescence microscopy (Model DMi8 automated, Leica Microsystems CMS GmbH, Germany).
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