Receptor recycling assays were performed as previously described (3 (link), 53 (link)). Briefly, primary microglia were plated on PDL-coated coverslips in 24-well plates at a density of 20,000 cells per well. Cells were maintained in DMEM with 10% FBS and 1% Pen/Strep and supplemented with 10 ng/mL M-CSF for 24 hours. Cells were then incubated in DMEM with 10% donkey serum (S30-M, Sigma-Aldrich) for 15 minutes at 37°C. Antibodies against CD36 (ab23680, Abcam) or TREM2 (AF1729, R&D Systems) were added to the cells in DMEM with 1% donkey serum for 1 hour at 37°C. Cells were then acid washed with cold DMEM at pH 2.0. Cells were cultured in DMEM with 10% donkey serum for 1 hour at 37°C and then incubated with fluorophore-conjugated secondary antibodies (Alexa Fluor 555 or Alexa Fluor 647, Invitrogen) in 1% donkey serum for 1 hour at 37°C. Cells were again acid washed with cold DMEM at pH 2.0 and washed with cold PBS. Cells were then fixed with 4% paraformaldehyde (PFA), washed with PBS, and mounted on glass slides. Similar experiments were performed on BV-2 cells plated on glass coverslips and pretreated with vehicle or 10 μM C3aRA (SB290157, Calbiochem). Fluorescent signal from vesicles containing recycled receptors were thresholded and the intensity of the fluorescence signal was determined by ImageJ (NIH) and normalized to WT or vehicle-treated cells.
Donkey serum
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
Donkey serum is a laboratory reagent derived from the blood of donkeys. It is commonly used as a blocking agent in various immunoassays and immunohistochemical techniques to reduce non-specific binding of antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Donkey serum, by Merck Group (11 mentions)
Donkey serum, by Jackson ImmunoResearch (8 mentions)
Triton x 100, by Merck Group (7 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (7 mentions)
Lsm 710, by Zeiss (6 mentions)
Lsm 880, by Zeiss (4 mentions)
Bovine serum albumin (bsa), by Merck Group (4 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (4 mentions)
Mouse anti gfap, by Merck Group (3 mentions)
Rabbit anti map2, by Merck Group (3 mentions)
Alexa fluor 488 donkey anti rabbit, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 594 donkey anti mouse, by Thermo Fisher Scientific (3 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (3 mentions)
Hoechst 33342, by Thermo Fisher Scientific (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Pan ck antibody, by Agilent Technologies (2 mentions)
Donkey anti rabbit igg h l, by Thermo Fisher Scientific (2 mentions)
Donkey anti mouse igg h l, by Thermo Fisher Scientific (2 mentions)
Normal donkey serum (nds), by Jackson ImmunoResearch (2 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (2 mentions)
56 protocols using donkey serum
1
Quantifying Receptor Recycling in Microglia
2
Immunofluorescent Detection of cFos in Tissue
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Sections were washed with phosphate-buffer saline 0.10 M triton X-100 (Sigma-Aldrich, Madrid, Spain; Cat# T9284) (1%) (PBST) and then incubated in a blocking solution (1.50% Donkey serum; Cat# D9663; Sigma-Aldrich, Madrid, Spain) in PBST for 30 min at room temperature. Subsequently, they were incubated with a polyclonal primary antibody, rabbit anti-cFos (1:500; Cat# 226003; Synaptic Systems, Göettingen, Germany) in PBST with Donkey serum (1.50%), in smooth agitation at 4°C for 24 h. After washing with PBST, sections were incubated with donkey anti-rabbit Alexa Fluor 555 conjugate (1:250; Cat # A-31572; Thermo Fisher Scientific, Madrid, Spain) in PBST with Donkey serum (1.50%) for 2 h at room temperature. Then, sections were rinsed with PBST and mounted with FluorSave reagent (Cat# 345789; Calbiochem; Millipore; Merck KGaA, Darmstadt, Germany).
3
Immunocytochemistry Visualization of Rhodopsin
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Immunocytochemistry (ICC) was performed using standard techniques. Briefly, 48 h post-transfection HEK293T cells were fixed with methanol-free 4% formaldehyde (Thermo Fisher Scientific) in PBS for 10 min at room temperature. Transfected cells were washed three times with 0.05% Tween-20 in PBS (PBS-T), permeabilised with 0.2% Triton X-100 in PBS for 5 min and blocked with 5% donkey serum (Sigma-Aldrich) in PBS-T for 30 min. Cells were incubated for 1 h with primary rabbit polyclonal anti-rhodopsin (ab3424, http://www.abcam.com/rhodopsin-antibody-ab3424.html ) and mouse monoclonal anti-1D4 antibodies (gift from Dr Jill Cowing, Institute of Ophthalmology, University College London), both diluted 1:1000 in 1% donkey serum in PBS-T. Cells were washed five times with PBS-T. Cells were incubated for 30 min with Alexa-488 and Alexa-568 conjugated secondary antibodies (A10037 and A21206, Life Technologies), 1:200 with 1% donkey serum in PBS-T before five washes with PBS-T. All steps were carried out at room temperature. Coverslips were mounted with ProLong Gold with DAPI (Life Technologies), left in the dark overnight and then stored at 4°C. Fluorescent images were acquired using an inverted confocal microscope (LSM 710, Carl Zeiss).
4
Immunocytochemical Characterization of CTCs
5
Immunohistochemical Analysis of MIS RII Expression
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Immunohistochemical analysis to detect MIS RII expression was performed using the Invitrogen Histostain Plus AEC kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Cultured AN3CA cells were harvested and 200 μl of a 1×105 cell/ml suspension was centrifuged with Cytospin (Thermo Electron Corp., Cheshire, UK) at 1000 rpm for 5 min and attached to Probe-on-plus-slides. These slides then were treated with 3% H2O2 for 5 min to eliminate endogenous peroxidase activity and washed three times with Tris-buffered saline with 0.1% Tween-20 (T-TBS). After treatment with donkey serum (Invitrogen) for 30 min to block non-specific protein binding, the slides were incubated with rabbit polyclonal anti-human MIS type II receptor antiserum (Massachusetts General Hospital, Boston, MA, USA) (11 (link)) at 4°C overnight. The slides were rinsed in T-TBS three times, and incubated with biotinylated anti-rabbit IgG (Invitrogen) for 30 min. After another three T-TBS rinses, the streptavidin HRP detection system (Invitrogen) was applied to the slides for 30 min to induce the biotin-avidin binding reaction. The slides were treated with 3-amino-9-ethylcarbazole (AEC, Invitrogen) for 10 min at room temperature, counterstained with hematoxylin, then mounted with glycerol gel.
6
Immunohistochemical Detection of Galectin-3 in Aortic Sinus
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Aortic sinus sections were fixed with 4% (v/v) paraformaldehyde (Thermo Scientific, J19943-K2) in PBS for 15 min, followed by washing with PBS three times, then slides were incubated with blocking buffer (2% donkey serum [Sigma, D9663] with 0.3% Triton X-100 [Sigma, T8787]) for one hour at room temperature. Sections were then incubated with anti-galectin 3 antibody (Invitrogen, 50-5301-82) at 1:200 diluted in dilution buffer (1% donkey serum with 0.1% Triton X-100) at 4 °C overnight. Sections were then washed in PBS three times and incubated with secondary antibody (Jackson ImmunoResearch, 712-585-153) at 1: 500 in dilution buffer for one hour at room temperature. After washing with PBS, sections were mounted with ProLongTM Gold antifade reagent with DAPI (Invitrogen, P36935), and images were taken under the fluorescent microscope under the same settings.
7
Immunofluorescence Staining of Cell Lines
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Commercial antigens were double stranded DNA (cat. D3664, SIGMA), insulin (I9278, SIGMA) and LPS (L6143, SIGMA). Confocal related reagents included antigen retrieval buffer low pH (cat. 00-4955-58, Invitrogen), donkey serum (cat. 017-000-121, Jackson ImmunoResearch), human Fc block (cat.584220, BD Phramingen) and Hoechst 33342 (cat.H3570, ThermoFisher), ProLong Gold anti-fade mounting solution (P36930, ThermoFisher). Commercial primary antibodies included, murine IgG anti-vimentin (clone V9, DAKO), rabbit IgG anti-enolase 1 (clone EPR 10863, Abcam) and rat IgG anti-DYKDDDDK (anti-FLAG, clone L5, Biolegend). Secondary antibodies (ThermoFisher) were Alexa Fluor 488 goat anti-rat IgG, Alexa Fluor 647 donkey anti-rabbit IgG, Alexa Fluor 568 anti-mouse IgG 568, Alexa Fluor 488 goat anti-human IgG and anti-human IgG HRP (Jackson ImmunoResearch).
8
Immunocytochemistry Analysis of Circulating Tumor Cells
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A three-color immunocytochemistry analysis (30 (link)) was adopted for immunofluorescence characterization of CTCs captured from blood samples collected from patients with NSCLC. The cells captured on Tz-grafted SiNWS were incubated with 0.05% Triton X-100 [in PBS (200 μl)] for 10 min. The captured cells were incubated overnight with a mixture of primary antibodies including Pan-CK antibody [rabbit, polyclonal, 1:100 (v/v); Dako] and anti-CD45 antibody [F10-89-4] [mouse, monoclonal, 1:400 (v/v); Abcam] in a PBS solution (200 μl) containing 2% normal donkey serum (Jackson ImmunoResearch) at 4°C. After washing with PBS three times, the captured cells were further incubated at room temperature for 1 hour with a mixture of secondary antibodies including donkey anti-rabbit IgG (H+L) [Alexa Fluor 488, 1:500 (v/v); Invitrogen] and donkey anti-mouse IgG (H+L) [Alexa Fluor 647, 1:500 (v/v); Invitrogen] in a PBS solution (200 μl) containing 2% donkey serum. After washing with PBS three times, the cells were treated with ProLong Gold Antifade Mountant with DAPI (Invitrogen). The substrates were then imaged with a fluorescence microscope (Nikon 90i). The captured CTCs were differentiated from background WBCs according to their unique staining pattern (CK+/CD45−/DAPI+) and intact nuclear morphology (WBCs were stained CK−/CD45+/DAPI+).
9
Hepatic Monocyte/Macrophage Immunofluorescence
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Hepatic monocyte/macrophages adhered to slides and were fixed by paraformaldehyde, followed by rinsing with PBS. The fixed monocyte/macrophages were permeabilized with 0.1% Triton X100 for 10 min and blocked with 5% donkey serum (100-151, Sacramento, CA, USA) for 1 h. Next, they were incubated with primary antibodies (CD14 MA1-23611, Invitrogen, Carlsbad, CA, USA), secreted phosphoprotein 1(SPP1) (ab8448, Abcam, Cambridge, UK), and CD68 (ab125212, Abcam, Cambridge, UK) overnight at 4 °C. After that, monocyte/macrophages were treated with secondary antibody (donkey anti-rabbit Alexa488, 1:300 dilution; donkey anti-mouse Alexa 568, 1:300 dilution; Invitrogen) for 30 min. Then, 4′,6-diamidino-2-phenylindole (DAPI;71010100, Biosharp, Hefei, China) was used to label nuclei. An anti-fluorescence attenuating agent (S2100, Solarbio, Beijing, China) was employed to mount the slides.
10
Immunocytochemistry Assay for LINGO-1 Expression
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