WT HeLa, HeLaΔCD164, or HeLaΔCD164 cells lentivirally transduced to express a recombinant WT (hCD164 PM) or N104Q (N104Q hCD164 PM), in which the NYxxL lysosomal targeting motif was deleted to increase plasma membrane (PM) expression to yield ΔCD164+hCD164 PM HeLa and ΔCD164+N104Q hCD164 PM HeLa, respectively, were used for immunofluoresence staining. The four different cell types were plated on 6-well plates, and 24 h later, cells were resuspended in PBS and 5 mM ethylenediamine tetraacetic acid. Cells were then washed twice in PBS and then fixed in 4% paraformaldehyde (PFA) for 20 min. Cells were washed twice on ice in PBS and then blocked in 1% BSA diluted in PBS (PBSA) for 30 min. Cells were then incubated on ice in a 1:500 dilution of sheep anti-human CD164 (R&D Systems No. AF5790) diluted in PBSA. Cells were washed three times in PBSA and then incubated with a 1:600 dilution of Donkey anti-sheep-488 (Thermo Fisher Scientific) in PBSA on ice. Cells were washed three times in PBSA and then subjected to flow cytometry analysis (FACS Canto), and the data were analyzed using the FlowJo software.
Donkey anti sheep 488
Manufactured by Thermo Fisher Scientific
Sourced in United States
Donkey anti-sheep-488 is a fluorescent-labeled secondary antibody used for the detection of sheep primary antibodies in various immunoassays and immunological techniques. It is conjugated with the fluorescent dye Alexa Fluor 488, which emits green fluorescence upon excitation.
Automatically generated - may contain errors
Lab products found in correlation
Donkey anti rabbit 647, by Thermo Fisher Scientific (2 mentions)
Goat anti rabbit 647, by Thermo Fisher Scientific (1 mentions)
Pierce goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Ahp500gt, by Bio-Rad (1 mentions)
Ab108338, by Abcam (1 mentions)
Ab184607, by Abcam (1 mentions)
Ab6663, by Abcam (1 mentions)
Ab48394, by Abcam (1 mentions)
Pierce goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Ab124762, by Abcam (1 mentions)
Ab64269, by Abcam (1 mentions)
Ab19166, by Abcam (1 mentions)
Opal 570, by Akoya Biosciences (1 mentions)
E0432, by Abcam (1 mentions)
T7451, by R&D Systems (1 mentions)
Donkey anti goat 568, by Thermo Fisher Scientific (1 mentions)
Donkey anti rat 488, by Thermo Fisher Scientific (1 mentions)
Gapdh rabbit polyclonal, by Abcam (1 mentions)
4 protocols using donkey anti sheep 488
1
Flow Cytometry Analysis of CD164 Variants
2
Antibody Panel for Western Blot and Immunofluorescence
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Unless otherwise noted, all chemicals were purchased from Fisher Scientific.
The following antibodies were used in this study: rabbit anti-AP-4 epsilon 1:400 for Western blots (612019; BD Transduction Labs); rabbit anti-ATG9A 1:200 for immunofluorescence and 1:1000 for Western blots (ab108338; Abcam); rabbit anti-LC3B 1:3000 for Western blots (ab48394; Abcam); mouse anti-LC3 1:200 for immunofluorescence (M152-3; MBL); HRP-conjugated anti-GFP 1:2000 for Western blots (ab6663; Abcam); rabbit anti-tepsin 1:500 (in-house; Genscript) and 1:1000 (Robinson Lab, Cambridge) for Western blots; sheep anti-TGN46 1:1000 for immunofluorescence (AHP500GT; Bio-Rad); mouse anti-alpha tubulin 1:3000 for Western blots (66031; Proteintech); mouse anti-Myc-tag 1:8000 for immunofluorescence and 1:6000 for Western blots (2276; Cell Signaling Technology); HRP-conjugated anti-6X His tag 1:8000 for Western blots (ab184607; Abcam); HRP-conjugated secondaries for Western blots, 1:5000: Pierce goat anti-rabbit IgG (31460; Thermo Fisher Scientific); Pierce goat anti-Mouse IgG (31430; Thermo Fisher Scientific); Fluorescent secondary antibodies for immunofluorescence, 1:500: goat anti-Rabbit 647 (A32733; Thermo Fisher Scientific), goat anti-Mouse 555 (A32727; Thermo Fisher Scientific), goat anti-Mouse 488 (A32723; Thermo Fisher Scientific), donkey anti-Sheep 488 (A11015; Thermo Fisher Scientific).
The following antibodies were used in this study: rabbit anti-AP-4 epsilon 1:400 for Western blots (612019; BD Transduction Labs); rabbit anti-ATG9A 1:200 for immunofluorescence and 1:1000 for Western blots (ab108338; Abcam); rabbit anti-LC3B 1:3000 for Western blots (ab48394; Abcam); mouse anti-LC3 1:200 for immunofluorescence (M152-3; MBL); HRP-conjugated anti-GFP 1:2000 for Western blots (ab6663; Abcam); rabbit anti-tepsin 1:500 (in-house; Genscript) and 1:1000 (Robinson Lab, Cambridge) for Western blots; sheep anti-TGN46 1:1000 for immunofluorescence (AHP500GT; Bio-Rad); mouse anti-alpha tubulin 1:3000 for Western blots (66031; Proteintech); mouse anti-Myc-tag 1:8000 for immunofluorescence and 1:6000 for Western blots (2276; Cell Signaling Technology); HRP-conjugated anti-6X His tag 1:8000 for Western blots (ab184607; Abcam); HRP-conjugated secondaries for Western blots, 1:5000: Pierce goat anti-rabbit IgG (31460; Thermo Fisher Scientific); Pierce goat anti-Mouse IgG (31430; Thermo Fisher Scientific); Fluorescent secondary antibodies for immunofluorescence, 1:500: goat anti-Rabbit 647 (A32733; Thermo Fisher Scientific), goat anti-Mouse 555 (A32727; Thermo Fisher Scientific), goat anti-Mouse 488 (A32723; Thermo Fisher Scientific), donkey anti-Sheep 488 (A11015; Thermo Fisher Scientific).
3
Multicolor Immunofluorescence Staining Protocol
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Sections were dewaxed 3 × 10 min in xylene and rehydrated in EtOH series (100%, 90%, 70%, 50%, and 30%) for a period of 2 min for each step. Antigen retrieval was performed in citric acid 0.01M at 90°C for 30 min. Blocking was achieved in PBS with 0.025% Tween20, 1% BSA, and 10% serum. After ON incubation at 4°C, the primary antibody was washed in PBT 0.025%, and a secondary antibody was incubated for 1 h at RT. After washing, the slides were mounted with fluoroshield. Primary antibodies were anti-acetylated tubulin 1/300 (Sigma-Aldrich, T7451), anti-Plunc1 1/200 (R&D systems, AF4274), and anti-K5 1/300 (Biolegend, PRB-160P), and secondaries were Goat anti-Mouse-568 1/500 (Invitrogen, A11004), Donkey anti-Sheep-488 1/500 (Invitrogen, A11015), and Donkey anti-Rabbit 647 1/500 (Invitrogen, A31573).
For the detection of PCNA 1/300 (Abcam, ab19166) and P63 1/400 (Abcam, ab124762), a secondary Goat anti-Rabbit biotin was used 1/800 (Dako, E0432) followed by Streptavidin-HRP (Abcam, ab64269). The color reaction was performed in TSA buffer (100 mM Borate buffer with 0.0003% hydrogen peroxidase) with Opal-570 1/300 (Akoya Bioscience, OP-001003) for 10min. DAPI was used to stain nuclei. For each genotype, N = 3.
For the detection of PCNA 1/300 (Abcam, ab19166) and P63 1/400 (Abcam, ab124762), a secondary Goat anti-Rabbit biotin was used 1/800 (Dako, E0432) followed by Streptavidin-HRP (Abcam, ab64269). The color reaction was performed in TSA buffer (100 mM Borate buffer with 0.0003% hydrogen peroxidase) with Opal-570 1/300 (Akoya Bioscience, OP-001003) for 10min. DAPI was used to stain nuclei. For each genotype, N = 3.
4
Immunofluorescence Antibody Optimization
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The Histone H3-S10P mouse monoclonal antibody (clone 6G3) was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA), whereas the H3-S28P rat monoclonal (clone HTA28) and the H4-R3me2 rabbit polyclonal antibody (catalog #39275) were obtained from Thermo Fisher Scientific (Rockford, IL, USA) and Active Motif (Carlsbad, CA, USA), respectively. The Histone H3-K4me3 rabbit monoclonal antibody (clone MC315) was purchased from Millipore, while the Histone H4-K16acetyl goat polyclonal antibody (catalog #sc-8662) was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The Msk2 rabbit polyclonal (catalog # ab42282), Lamin-B1 rabbit polyclonal (catalog #ab16048) and Gapdh rabbit polyclonal (catalog #ab9485) antibodies were obtained from Abcam (Cambridge, MA, USA).
The Alexa Fluor donkey anti-mouse 405 antibody (custom synthesis), donkey anti-rat 488, donkey anti-sheep 488, donkey anti-goat 568 and donkey anti-rabbit 647 antibodies were purchased from Invitrogen (Carlsbad, CA, USA) for confocal imaging analysis. As controls, the mouse, rat, rabbit and goat IgG isotype antibodies were obtained from Vector Laboratories (Burlingame, CA, USA).
The Alexa Fluor donkey anti-mouse 405 antibody (custom synthesis), donkey anti-rat 488, donkey anti-sheep 488, donkey anti-goat 568 and donkey anti-rabbit 647 antibodies were purchased from Invitrogen (Carlsbad, CA, USA) for confocal imaging analysis. As controls, the mouse, rat, rabbit and goat IgG isotype antibodies were obtained from Vector Laboratories (Burlingame, CA, USA).
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