Hela cells (7 × 104 cells) were seeded on the coverslips and fixed with permeabilization solution. After fixation, the cells were then incubated with mouse anti-Chikungunya virus antibody (Abcam, 1:25 dilution) and rabbit anti-vimentin antibody (CST, 1:100 dilution), followed by incubation with Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) (abcam, 1:200 dilution) and Donkey Anti-Rabbit IgG H&L (Alexa Fluor® 568) (abcam, 1:200 dilution). Cell nuclei were stained with 4′,6′-diamidino-2-phenylindole (DAPI) (Invitrogen). The samples were viewed using Laser confocal microscopy system (Leica, TCS SP5) under an oil mirror. The scale bars are 5 μm and resolution on imaging data is 512 × 512.
Donkey anti rabbit igg h l
Manufactured by Abcam
Sourced in United States, United Kingdom
Donkey anti-rabbit IgG H&L is a secondary antibody that binds to the heavy and light chains of rabbit immunoglobulin G (IgG) antibodies. It is used in various immunoassays and detection techniques to identify and quantify rabbit primary antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Donkey anti mouse igg h l, by Abcam (3 mentions)
Goat anti rat igg h l, by Abcam (2 mentions)
Donkey anti mouse igg h l, by Thermo Fisher Scientific (2 mentions)
Sytox orange nucleic acid stain, by Thermo Fisher Scientific (2 mentions)
Rabbit anti lyz, by Agilent Technologies (2 mentions)
Rabbit anti olfm4, by Cell Signaling Technology (2 mentions)
Recombinant rabbit anti aldolase b aldolase c, by Abcam (2 mentions)
Mouse anti human krt20, by Agilent Technologies (2 mentions)
Mouse anti chr a, by Santa Cruz Biotechnology (2 mentions)
Rat anti e cadherin, by Santa Cruz Biotechnology (2 mentions)
Cd24 monoclonal antibody, by Thermo Fisher Scientific (2 mentions)
Goat anti rabbit igg h l alexa fluor 405, by Abcam (2 mentions)
Wga conjugated to cf488a, by Biotium (2 mentions)
Reddot1 far red nuclear stain, by Biotium (2 mentions)
Mouse anti ki67, by BD (2 mentions)
Donkey anti goat igg h l, by Abcam (2 mentions)
Goat anti mouse igg h l, by Abcam (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Lsm 880, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
19 protocols using donkey anti rabbit igg h l
1
Chikungunya Virus Localization in Hela Cells
2
Quantitative Western Blot Analysis of Lung Tissue
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Cells or dissected mouse lung tissue samples were lysed in ice-cold RIPA lysis buffer with protease inhibitors. Protein concentrations were determined using a BCA Protein Quantitative Analysis Kit (Fudebio-tech) after which protein samples were separated by 8–12% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore). The membranes were then incubated at room temperature for 1 h in TBST containing 5% BSA. After blocking, the membranes were incubated with primary antibodies for 24 h at 4°C.The following primary antibodies were used: anti-Fibronectin (Abcam, ab268020); anti-Collagen I (affinity, AF7001); anti-alpha smooth muscle (Abcam, ab5694); anti-PFKFB3 (Abcam, ab181861); anti-Beta actin (proteintech, 66009-1-Ig); anti-Hexokinase 2 (proteintech, 22029-1-AP); anti-PKM2 (Proteintech, 15822-1-AP); anti-LDHA (Proteintech, 19987-1-AP); anti-LDHB (Proteintech, 14824-1-AP); and anti-PCBP3 (Abcam, ab154252). Then, the membranes were washed three times with TBST and incubated with donkey anti-rabbit IgG H&L (Abcam, ab175772) for 1 h at room temperature. The membranes were developed using the ECL method according to the manufacturer’s instructions (Millipore) and detected on a GeneGnome XRQ chemiluminescence imaging system (Syngene). ImageJ was used to calculate the relative density of proteins.
3
Immunofluorescence Staining of Cardiomyocytes
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Heart section (5 μm) were subject to deparaffinization and rehydration followed by antigen retrieval by heating the slides in 10 mM Citrate buffer (pH 6.0) at 95°C for 10 min. For cardiomyocytes, NRCMs cultured on glass coverslips were fixed in paraformaldehyde for 15 min at room temperature. After washing for three times with PBS each for 5 min, cardiomyocytes and heart sections were permeabilized with 0.5% (vol./vol.) Triton X‐100 for 15 min and washing for three times with PBS each for 5 min then blocked with 5% (vol./vol.) bovine serum albumin (BSA, Sigma‐Aldrich, B2064) for 1 h at room temperature. Next, the samples were incubated primary antibodies at 4°C overnight. After washing, samples incubated secondary antibodies. Finally, DAPI was used to label the cell nuclei. Images were acquired with a Leica SP8 confocal microscopy (Leica TCS SP8, Wetzlar, Germany).
The primary antibodies used are as follows: FGF6 (Santa Cruz, sc‐373927), c‐TNT (ABclonal, A4914), Ki‐67 (D3B5) Rabbit mAb (Alexa Fluor® 647 Conjugate) (CST, 12075), YAP (CST, 14074), Aurora B (Abcam, ab2254).
The secondary antibodies used are as follows: Goat Anti‐Mouse IgG H&L (Alexa Fluor® 488) (Abcam, ab150113), Goat Anti‐Mouse IgG H&L (Alexa Fluor® 647) (Abcam, ab150115), Goat Anti‐Rabbit IgG H&L (Alexa Fluor® 488) (Abcam, ab150077), Donkey Anti‐Rabbit IgG H&L (Alexa Fluor® 647) (Abcam, ab150075).
The primary antibodies used are as follows: FGF6 (Santa Cruz, sc‐373927), c‐TNT (ABclonal, A4914), Ki‐67 (D3B5) Rabbit mAb (Alexa Fluor® 647 Conjugate) (CST, 12075), YAP (CST, 14074), Aurora B (Abcam, ab2254).
The secondary antibodies used are as follows: Goat Anti‐Mouse IgG H&L (Alexa Fluor® 488) (Abcam, ab150113), Goat Anti‐Mouse IgG H&L (Alexa Fluor® 647) (Abcam, ab150115), Goat Anti‐Rabbit IgG H&L (Alexa Fluor® 488) (Abcam, ab150077), Donkey Anti‐Rabbit IgG H&L (Alexa Fluor® 647) (Abcam, ab150075).
4
Multicolor Immunostaining Protocol
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The primary antibodies used in this study are as follows: rabbit anti-Lyz (1:800; Dako, #A0099), rabbit anti-Olfm4 (1:500; Cell Signaling Technology, #39141), recombinant rabbit anti-aldolase B + aldolase C (1:300; Abcam, #ab75751), mouse anti-human KRT20 (1:500; Dako, #M701929-2), mouse anti–Chr-A (1:50; Santa Cruz Biotechnology, #sc-393941), rat anti–E-cadherin (1:400; Santa Cruz Biotechnology, #sc-59778), CD24 Monoclonal Antibody (1:200; Thermo Fisher Scientific, #17-0242-80), and mouse anti-Ki67 (1:200; BD Biosciences, 550609). The secondary antibodies used in this study are as follows: Goat Anti-Rabbit IgG H&L (Alexa Fluor 405) preadsorbed (1:1000; Abcam, #ab175654), Goat Anti-Rat IgG H&L (Alexa Fluor 555) preadsorbed (1:1000; Abcam, #ab150166), Donkey Anti-Mouse IgG H&L (Alexa Fluor 647) (1:500; Thermo Fisher Scientific, #A31571), and Donkey Anti-Rabbit IgG H&L (Alexa Fluor 405) preadsorbed (1:1000; Abcam, #ab175649). The dyes used in this study are as follows: WGA conjugated to CF488A (5 μg/ml; Biotium), RedDot1 Far-Red Nuclear stain (1:200; Biotium), and SYTOX Orange Nucleic Acid Stain (1:5000; Thermo Fisher Scientific, #S11368). The order of staining, optimized to ensure good staining quality for all cell types, was based on the staining quality and stripping difficulty of each antibody (fig. S1G).
5
Evaluating hESC-OP9 Coculture Differentiation
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To analyze the differentiation efficiency of the coculture system, hESCs/OP9 coculture cells were treated with 0.25% trypsin (Gibco, USA) for 20 min at 37 °C on days 8, 10 and 12. trypsinization was terminated by adding a coculture medium. Co-culture cells were harvested by centrifugation, and washed twice by washing buffer (PBS supplemented by 5% FBS) [16 ]. These cells were immediately incubated with APC-human TRA-1–85 (R&D, USA), PE-Cy5.5 mouse anti-human CD34 (Becton Dickinson Company, USA) for 30 min at 4 °C. After staining, these cells were washed with washing buffer, centrifuged, and resuspended with moderate washing buffer, followed by flow cytometric analysis (BD FACSAria™ II sorter (BD Biosciences, USA). Data were analyzed with FlowJo version 7.6.1 (FlowJo, LLC). For negative control, APC mouse anti-human CD34 (Becton Dickinson Company, USA) was used to HN14, while OP9 cells labeled anti-CD34 [EP373Y] (Abcam, USA) and used Donkey Anti-Rabbit IgG (H&L) (Abcam, USA) as the secondary antibody.
6
Neonatal Cardiomyocyte Proliferation Assay
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Cell proliferation was determined by Ki67 immunostaining. Neonatal CMs were seeded at 1×104 cells/well in 24-well plates and cultured under normoxic conditions (37°C, 5% CO2, 5% O2) for 7 days. The proliferating cells were detected by double staining with Ki67 and TnI. Briefly, the cells were fixed in 4% paraformaldehyde, blocked with 5% BSA after 10 min of permeabilization, and incubated with primary antibodies [Ki67 (ab16667), TnI (ab30807) both from Abcam] overnight followed by the respective fluorescent conjugated secondary antibodies [donkey anti-rabbit IgG H&L (DyLight 488; ab96891); and donkey anti-goat IgG H&L (DyLight 550; ab96932); both from Abcam]. After 3 washes with PBST, the cell nuclei were stained with Hoechst 33258 pentahydrate 1 µg/ml (Invitrogen). Fluorescence images were then acquired using a Leica fluorescence microscope.
7
Immunofluorescent Staining of Bile Ducts
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Liver lobes were fixed in 4% paraformaldehyde overnight in 4°C. Then, lobes were placed in 30% sucrose in phosphate‐buffered saline (PBS) until tissue sinks (6–12 h) and embedded in OCT.
Immunofluorescent staining was performed on 13 µm frozen liver sections. Slides were incubated in cold acetone for 7 min and dried at room temperature. Following washing, slides were blocked with 1% bovine serum albumin for 1 h at room temperature. Samples were stained overnight at 4°C with anti‐CK19 (Abcam) to identify bile ducts. Slides were washed 3 times for 5 min in PBS−/− and stained with secondary antibody (Donkey Anti‐Rabbit IgG H&L, 1:1000, Alexa Fluor 647; Abcam) for 1 h at room temperature.
Eventually the slides were cover slipped with Fluorescent Mounting Medium with 4,6‐diamidino‐2‐phenylindole (DAPI; GBI Labs). Images were taken with ZEISS Confocal Microscope (MicroImaging GmbH, ZEISS). Processing was performed with ZEN 2010 software.
Immunofluorescent staining was performed on 13 µm frozen liver sections. Slides were incubated in cold acetone for 7 min and dried at room temperature. Following washing, slides were blocked with 1% bovine serum albumin for 1 h at room temperature. Samples were stained overnight at 4°C with anti‐CK19 (Abcam) to identify bile ducts. Slides were washed 3 times for 5 min in PBS−/− and stained with secondary antibody (Donkey Anti‐Rabbit IgG H&L, 1:1000, Alexa Fluor 647; Abcam) for 1 h at room temperature.
Eventually the slides were cover slipped with Fluorescent Mounting Medium with 4,6‐diamidino‐2‐phenylindole (DAPI; GBI Labs). Images were taken with ZEISS Confocal Microscope (MicroImaging GmbH, ZEISS). Processing was performed with ZEN 2010 software.
8
Calcium Silicide-Iodine Electrode Fabrication
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Calcium silicide (CaSi2, 95%, Gelest), iodine (I2, 99.99%, Sigma‐Aldrich), acetonitrile (CH3CN, 99.8%, Sigma‐Aldrich), 1‐methyl‐2‐pyrrolidinone (NMP, 98%, Sigma‐Aldrich), double distilled water (Milli‐Q system, Millipore, USA), hexadecyltrimethylammonium chloride (CTAC, 99%, Adamas), triethylamine (TEA, 99%, Adamas), tetraethyl orthosilicate (TEOS, SiO2 iOrolidinGreagent), ethanol absolute (C2H6O, ≥ 99.7%, Shanghai Lingfeng Chemical Reagent Co. Ltd), hydrochloric acid (HCl, 36.0–38.0%, Shanghai Lingfeng Chemical Reagent Co. Ltd), and RP hydrochloride monohydrate (Adamas, 99%) were used. Antibodies which were used included Iba‐1 (WAKO, 019–19741), GFAP (Abcam, ab53554), TRPV1 (Abcam, ab5566), c‐Fos (CST, 2250S), Donkey Anti‐Rabbit IgG H&L (Abcam, ab150073), and Donkey Anti‐Goat IgG H&L (Abcam, ab150132). All chemicals used in slice preparation and electrophysiological recording were purchased from Sigma‐Aldrich.
9
Melanocyte Immunofluorescence Staining
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When the primary epidermal cells reached 80% confluence, they received differential trypsinization to obtain MCs, which were seeded in six‐well plates containing M254 medium and then cultured at 37°C with 5% CO2. The culture medium was changed every 2 days. In order to perform immunofluorescence staining, the MCs were subcultured in 24‐well plates at a density of 1 × 105 cells per well. The adherent cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X‐100. The primary antibodies (HMB45 [1:500, cat. no. ab787, Abcam, UK] and TYR [1:1000, cat. no. ab738, Abcam]) and their corresponding fluorescent (Alexa Fluor 647) conjugated secondary antibodies, isotype‐matched donkey anti‐mouse immunoglobulin G (IgG, 1:400, cat. no. ab150105, Abcam), and donkey anti‐rabbit IgG H&L (1:400, cat. no. ab150075, Abcam) were used. Counterstaining with 4', 6‐diamidino‐2‐phenylindole (DAPI) (1:100, cat. no. MA0127, Meilunbio, Dalian, China) was performed to identify nuclei. Images were taken using a fluorescence microscope (Olympus, Japan).
10
Co-culture Immunofluorescence Assay
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To evaluate the culturing performance and layer-arrangement of NHDFs and HaCaTs, after progressing co-culture of the two cells on the scaffold, immunofluorescence staining using anti-fibronectin (FN, Abcam, Cambridge, UK), a marker specific for fibroblasts, and anti-cytokeratin-14 (CK14, Abcam, Cambridge, UK), a marker for identifying keratinocytes, was performed. After fixation of the cultured scaffolds with 4% paraformaldehyde (PFA, Biosesang, Seongnam, Korea), blocking was achieved by incubating the cells for 60 min in 1% normal goat serum (Vector laboratories, Burlingame, CA, USA) in PBS/0.3% Triton X-100 (Sigma, St. Louis, MO, USA). The cells were incubated overnight at 4 °C with primary antibodies (Abcam, Cambridge, UK) against fibronectin or cytokeratin-14 at a dilution of 1:200. The cells were then labeled with secondary antibodies of donkey anti-mouse IgG H&L (anti-mouse, Abcam, Cambridge, UK) or donkey anti-rabbit IgG H&L (anti-rabbit, Abcam, Cambridge, UK) at a dilution of 1:500 for 1 h at 37 °C. After washing with Tris-buffered saline, twice, the scaffolds were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma, St. Louis, MO, USA) and images were acquired using a confocal microscope (LSM-710, Zeiss, Oberkochen, Germany).
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