After 24 h, the cells were incubated with either 1% DMSO or compounds for 2 h, and then treated as following steps: 1) fixated with 4% paraformaldehyde for 10 min, 2) permeabilized with PBS/0.1% Triton-X for 10 min, 3) blocked with donkey serum blocking buffer [10% donkey serum (Solarbio) in PBS] at a period of 60 min, washed the cells with PBS three times between each steps. The anti-Pan-RAS mouse monoclonal antibody (Calbiochem # OP40-100UG; 1:40) and anti-PDEδ antibody (Genetax # GTX109240, 1:500) were mixed and incubated in blocking buffer overnight at 4 °C. Afterwards, the cells were washed with PBS three times. The secondary antibody, Dylight 649 nm goat anti-Mouse antibody (Abbkine; 1:1000) and Alexa 488 nm goat anti-rabbit antibody (abcam #ab150077, 1:1000) was incubated for another 2 h. The cells were washed three times with PBST, and the images were collected by confocal microscopy (Leica TCS SP5 equipped with Argon-Heli-umNenon laser).
Donkey serum
Manufactured by Solarbio
Sourced in China
Donkey serum is a raw material commonly used in cell culture and immunological applications. It is collected from healthy donkeys and processed to meet laboratory standards.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Beyotime (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Abcam (3 mentions)
Triton x 100, by Merck Group (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (2 mentions)
Antifade mounting medium, by Solarbio (2 mentions)
Fluorescence microscope, by Leica (2 mentions)
Fluorescence microscope, by Olympus (2 mentions)
Ab6994, by Abcam (2 mentions)
Hoechst 33342, by Merck Group (2 mentions)
Ab150077, by Abcam (2 mentions)
Triton x 100, by Beyotime (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Desmin, by Abcam (1 mentions)
Fv1000, by Olympus (1 mentions)
Confocal fluorescence microscopy, by Zeiss (1 mentions)
Alexa fluor 488 conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Phosphate buffered saline (pbs), by Sangon (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
31 protocols using donkey serum
1
Immunostaining of RAS and PDEδ Proteins
2
Cardiomyogenic Differentiation of MSCs
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Induction of MSC differentiation into cardiomyocytes was performed as previously described with slight modifications [27 (link)]. In brief, GFP-expressing MSCs were co-cultured with isolated neonatal rat cardiomyocytes at a ratio of 5000:5000 cells on a slide for 1 week, followed by immunofluorescence analysis. The medium was replenished every 3 days.
Differentiated MSCs were characterized by immunocytochemistry analysis as previously described [32 (link)]. In brief, cells were fixed in 4% paraformaldehyde for 10 min, permeabilized with 0.5% Triton X-100 for 20 min, and blocked in 10% donkey serum (Beijing Solarbio Science & Technology Co., Ltd.). Cells then were incubated with antibodies against cTnT (1:400, Abcam) and desmin (1:100, Abcam) at 4 °C overnight. The cells were then further incubated with the appropriate secondary antibodies (Abcam) diluted at 1:100 for 1 h at room temperature, followed by another incubation with DAPI (Beijing Solarbio Science & Technology Co., Ltd.). Untreated rat MSCs and adult cardiomyocytes were used as negative and positive controls, respectively. The results of immunostaining were observed under fluorescence microscopy (Olympus FV1000).
Differentiated MSCs were characterized by immunocytochemistry analysis as previously described [32 (link)]. In brief, cells were fixed in 4% paraformaldehyde for 10 min, permeabilized with 0.5% Triton X-100 for 20 min, and blocked in 10% donkey serum (Beijing Solarbio Science & Technology Co., Ltd.). Cells then were incubated with antibodies against cTnT (1:400, Abcam) and desmin (1:100, Abcam) at 4 °C overnight. The cells were then further incubated with the appropriate secondary antibodies (Abcam) diluted at 1:100 for 1 h at room temperature, followed by another incubation with DAPI (Beijing Solarbio Science & Technology Co., Ltd.). Untreated rat MSCs and adult cardiomyocytes were used as negative and positive controls, respectively. The results of immunostaining were observed under fluorescence microscopy (Olympus FV1000).
3
Immunofluorescence Staining of p65 in PDLSCs
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PDLSCs treated with LPS, LPS + shNC, or LPS + shEZH2 were seeded on glass bottom culture dishes in a 24-well plate, fixed with 4% paraformaldehyde (Sigma) for 10 min, and perforated with 0.2% Triton-X100 for 5 min. After blocking with 10% donkey serum (Solarbio, Beijing, China) for 20 min and washing by cold 1 × PBS (Sangon Biotech, Shanghai, China), the cells were incubated with p65 (Abcam, MA, USA) at 4°C overnight. The culture dishes were incubated with AlexaFluor488-conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA, USA) for 2 h. The nuclei of cells were stained with DAPI (Solarbio, Beijing, China) for 10 min. Confocal fluorescence microscopy (Zeiss Germany, Germany) was used to capture fluorescence confocal images.
4
Immunocytochemistry of Neural Cell Types
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The following types of cells were plated separately at a density of 105 cells/cm2 and cultured for 24 h: microglia, neurospheres, proliferative NSPCs, and differentiated NSPCs. Cells were fixed with 4% paraformaldehyde (pH 7.2) for 30 min. Slices containing the hippocampus and culture cells were permeabilized with 0.5% Triton X‐100 in PBS for 15 min, blocked in 10% donkey serum (Solarbio) for 2 h, and then incubated overnight at 4°C with the following primary antibodies as listed in Table S3 . Slices or cells were washed three times with PBS and then incubated for 2 h at room temperature with DyLight 549‐ or DyLight 488‐conjugate secondary antibodies (both 1:300; Jackson ImmunoResearch). Finally, cells were incubated for 5 min with 4′, 6‐diamidino‐2‐phenylindole (DAPI; 1:10,000, Roche) and imaged using a fluorescence microscope (Olympus IX 73).
5
Histological Analysis of Tendon-Bone Junction
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The tendon-bone junction tissue was fixed, decalcified, and sectioned (7 μm), and routine hematoxylin-eosin (HE) and Safranin O-Fast Green (Solarbio, China) staining were performed according to the manufacturer’s protocol. The immunofluorescence staining steps were as follow. Sections were permeabilized in 0.3% Triton X-100 (Beyotime, China) for 15 min, blocked in 5% donkey serum (Solarbio, China) for 30 min, and then incubated at 4°C overnight with the following primary antibodies: inducible nitric oxide synthase (iNOS) antibody (Cell Signaling Technology, 13120S, 1: 400), Arginase-1 (Arg1) antibody (Cell Signaling Technology, 93668T, 1: 50), collagen II (Col II) antibody (Abcam, ab34712, 1: 200), proliferating cell nuclear antigen (PCNA) antibody (Cell Signaling Technology, 13110T, 1: 500), and CD146 antibody (Abcam, ab75769, 1: 200). The next day, the sections were incubated with fluorescence secondary antibody (Cell Signaling Technology, 4412S, 1: 500) at room temperature for 2 h and counterstained with DAPI (Beyotime, China). Finally, the sections were observed, and photos were taken under a laser scanning confocal microscope (LSCM; Zeiss, Germany).
6
Immunofluorescence of Respiratory Syncytial Virus
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Immunofuorescence was carried out using lung tissues. Lung sections were prepared for immunofuorescence after deparafnized, dehydrated and antigen retrieval. Next, the fixed tissue samples were mounted on the cover glass, blocked with donkey serum (Solarbio, Beijing, China) and probed with RSV-antibody. After rinsing thrice with PBS, the fixed tissue sections were incubated with the secondary antibody at 37 °C for 50 min in the dark. Afterwards, the cell nuclei were stained with 4′,6-diamidino-2-phenylindole at 37˚C for 10 min in the dark. Eventually, the sectioned tissues were covered with an anti-fade mounting medium for fuorescence microscopy (Olympus).
7
Immunofluorescence and TUNEL Staining of Zebrafish Embryos
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For immunofluorescence staining, the embryos were anesthetized and then fixed using 4% paraformaldehyde. After washing three times with PBS-T, the embryos were incubated in the antigen retrieval solution (Beyotime Biotechnology, China, #P0088) for 15 min at 98°C. Non-specific binding was then blocked with 10% donkey serum (Solarbio, China, #SL050) in PBS-T. Next, specific primary antibodies against GFP (Abcam, #ab13970) and cleaved caspase-3 (CST, #9664), 5-bromo-2′-deoxyuridine (BrdU) (Sigma, #B5002) or SOX2 (Abcam, #ab97959) were added, and secondary antibodies were used to detect the primary antibodies.
TdT-mediated dUTP nick end labeling (TUNEL) assay was performed according to the manufacturer’s instructions (Alexa Fluor 640, cat#: 40308ES20, YEASEN Biotech Co. Ltd) to detect cell death in the HCs of neuromast. In brief, the embryos were anesthetized and then fixed using 4% paraformaldehyde. After washing three times with PBST, 20 μg/mL proteinase K (Roche) was used to treat the embryos. Next, Alexa Fluor 640-12-dUTP Labeling Mix was applied to label the apoptotic cells for at least 3 h. DAPI was applied to label the nucleus.
Images were taken with a Nikon confocal microscope A1R at 40× magnification and were analyzed by Nikon A1R NIS Elements. Exposure settings were adjusted to minimize oversaturation.
TdT-mediated dUTP nick end labeling (TUNEL) assay was performed according to the manufacturer’s instructions (Alexa Fluor 640, cat#: 40308ES20, YEASEN Biotech Co. Ltd) to detect cell death in the HCs of neuromast. In brief, the embryos were anesthetized and then fixed using 4% paraformaldehyde. After washing three times with PBST, 20 μg/mL proteinase K (Roche) was used to treat the embryos. Next, Alexa Fluor 640-12-dUTP Labeling Mix was applied to label the apoptotic cells for at least 3 h. DAPI was applied to label the nucleus.
Images were taken with a Nikon confocal microscope A1R at 40× magnification and were analyzed by Nikon A1R NIS Elements. Exposure settings were adjusted to minimize oversaturation.
8
Immunofluorescence Staining of Lung Tissue and HUVECs
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Immunofluorescence was performed with pulmonary tissues and HUVECs. The 4 μm sections of lung tissue were deparaffinized with xylene and dehydrated in a gradient series of ethanol. Furthermore, after antigen retrieval, the sections were prepared for immunofluorescence. Treated HUVECs were fixed in 4% paraformaldehyde to continue the experiment. The tissue sections and fixed cells on cover glass were further blocked with donkey serum (Solarbio, Beijing) and incubated with an HSPG2-antibody (1:200). After being washed three times with PBS, the sections and fixed cells were incubated with a second antibody (Alexa Fluor® 594) (1:200) at 37 for 1 h and further incubated with DAPI (Abcam) for 5 min. Finally, we sealed the stained sections and cells with an antifade mounting medium (Solarbio, Beijing, China) and observed them with a fluorescence microscope (Leica).
9
Isolation and Characterization of Neural Progenitor Cells
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Rats were sacrificed with an overdose of pentobarbital sodium (100 mg/kg, intraperitoneal injection) and the NPCs, AFCs and DCs were prepared as previously described (15 (link),17 (link),18 (link)). The cells used in this experiment were at the third passage (P3). The cells type and purity were identified by immunofluorescence staining for collagen-II and aggrecan. The cells were blocked with 5% donkey serum (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) for 30 min at room temperature and incubated overnight with rabbit anti-rat collagen II (1:200; cat. no. ab188570; Abcam, Cambridge, MA, USA) and rabbit anti-rat aggrecan (1:200, cat. no. 13880-1-AP, ProteinTech Group, Inc., Chicago, IL, USA) primary antibodies at 4°C. Subsequently, following an incubation with goat anti-rat secondary antibody (1:500, cat. no. M21003, Abmart, Inc., Shanghai, China) for 2 h at room temperature, nuclei were stained DAPI. The observation was carried out with a fluorescence microscope (Olympus Corp., Tokyo, Japan). Transplanted NPCs expressing GFP (GFP-NPCs) were isolated from GFP transgenic SD rats and used to identify the location of the transplanted cells.
10
Gelatin-Based Hydrogel Synthesis and Characterization
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Gelatin was purchased from Macklin (Shanghai, China). Ethylenediamine, 1,3,5-triformylbenzene (TFB), GMA, and polyethylene glycol (PEG) 10,000 were purchased from Sigma-Aldrich (St Louis, MO, USA). Thermo Fisher Scientific (New Jersey, USA) supplied 2,2-dimethoxy-2-phenylacetophenone (DMPA). Ethanol and NaBH4 were obtained from the Tianjin Kermel Chemical Reagent plant (Tianjin, China). Methanol and acetonitrile (ACN) were purchased from Merck (Darmstadt, Germany). The Zamboni solution was purchased from Tiandz (Beijing, China). Donkey serum, Triton X-100, Tween-20, CollagenaseI, and 1x phosphate buffer solution (1x PBS) were obtained from Solarbio (Beijing, China). The syringe (1 mL) was purchased from Hvsco (Beijing, China). Water was purified using a Unique-R20 purification system (Xiamen, China). All reagents and chemicals were at least of analytical grade.
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