Embryonic thymus was dissected and fixed in 4% PFA at 22°C for 30 min before soaking overnight at 4°C in 30% sucrose PBS solution. Samples were embedded in gelatine, flash-frozen and sectioned to 20 μm on Superfrost Plus slides (Thermo Fisher Scientific). For IL-33 staining, sections were permeabilized, stained overnight at 4°C with anti-IL-33 (AF3626, R&D Systems), then for 1 h at 22°C with donkey anti-goat IgG AF647 antibody (ab150131, Abcam). Sections were mounted using Prolong Gold (Thermo Fisher Scientific). Images were obtained with a 20x objective lens using a Zeiss 780 inverted confocal microscope with Zen software (version 14.0.19.201, configuration 6.00.00). Sequential laser scanning was used to minimize spillover between fluorescent proteins.
Ab150131
Manufactured by Abcam
Sourced in United Kingdom
Ab150131 is an antibody product from Abcam. It is a primary antibody.
Automatically generated - may contain errors
Lab products found in correlation
Ab150073, by Abcam (4 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (4 mentions)
Sytox green, by Thermo Fisher Scientific (3 mentions)
Af3626, by R&D Systems (2 mentions)
780 inverted confocal microscope, by Zeiss (2 mentions)
Prolong gold, by Thermo Fisher Scientific (2 mentions)
Ab150116, by Abcam (2 mentions)
Ab104139, by Abcam (2 mentions)
Ab150105, by Abcam (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
A21206, by Thermo Fisher Scientific (2 mentions)
Af2225, by R&D Systems (1 mentions)
Anti cd8 bv510, by BioLegend (1 mentions)
Anti ifn γ bv 421, by BioLegend (1 mentions)
Ab75472, by Abcam (1 mentions)
Sc 7312, by Santa Cruz Biotechnology (1 mentions)
Af 4519, by R&D Systems (1 mentions)
Glass coverslips, by NEST Biotechnology (1 mentions)
Af3628, by R&D Systems (1 mentions)
Image pro plus 6, by Olympus (1 mentions)
18 protocols using ab150131
1
Immunostaining of Embryonic Thymus
2
Imaging Innate and Adaptive Immune Responses
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Procedure has been performed as previously described69 (link). Briefly, WT mice were infected with 104 colony-forming units LmOVA. Mice were given 250 μg brefeldin A by i.p. injection, 6 h before being sacrificed. After 24 h, mice were euthanised, and the spleens were removed, fixed in 4% PFA and cryopreserved in OCT. Serial sections (30 µm in thickness) of frozen spleens were stained overnight at +4 °C in a humidified chamber with the following Abs: anti-B220 PB (1:300; 103230, Biolegend), anti-CD169 Alexa647 (1:300; 142407, Biolegend), anti-CD8 BV510 (1:100; 100751, Biolegend), anti-IFNγ BV421 (1:200; 505829, Biolegend), anti-I-A/I-E biotin (1:200; 107603, Biolegend), or anti-NKp46 (1:200; AF2225, R&D). For secondary detection, sections were then washed and incubated with Streptavidin-Cy3 (1:200; 016-160-084, Jackson Immunoresearch) and anti-goat IgG A647 (1:500; ab150131; Abcam) for 3 h at RT. All sections were analysed by confocal microscopy. Quantification of the interaction between DCs and IFNγ-producing cells was performed manually. DCs were randomly selected in the IFNγ-rich area. For each DC, the number of IFNγ-producing CD8 T cells and the number of IFNγ-producing NK cells was recorded and averaged.
3
Quantifying Integrin and Glypican-1 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
EAhy926 cells were plated onto glass coverslips (NEST Biotechnology) and serum-starved for 12 h after they entered logarithmic growth phase. Control cells were incubated in serum-free culture medium, HM-3 treatment groups were incubated with 4.5 or 17.8 μM HM-3 for 1 h in serum-free cell culture medium, enzyme treatment groups were incubated with PI-PLC (0.5 U/ml) at 30 °C for 30 min before HM-3 treatment. Cells were washed three times with PBS, fixed with 4% paraformaldehyde at 4 °C for 10 min and then blocked with 5% BSA. Cells were then incubated with anti-integrin αvβ3 (SC-7312, Santa Cruz Biotech), or anti-integrin α5β1 (ab75472, Abcam) and anti-Glypican-1 (AF-4519, R&D Systems) antibodies at 4 °C overnight. After washing twice with PBS, cells were incubated with fluorescently labeled secondary antibodies (Alexa Fluor 647 or 488 labeled ab150131 or ab150073, Abcam). Nuclei were counterstained with Hoechst 33342 (H1399, Thermo Fisher Scientific). Images were captured with an OLYMPUS workstation and analyzed with Image J software.
4
Choroid Tissue Immunohistochemistry Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Choroid film and immunohistochemical analyses were performed as previously described (31 (link)). The primary antibodies used were anti-RGS1 (1:1,000, Abcam cat# ab117077, RRID: AB_2916361) and anti-platelet endothelial cell adhesion molecule-1 (1:200, R&D Systems cat# AF3628, RRID: AB_2161028). The secondary antibody included horseradish peroxidase (HRP)-conjugated anti-goat antibody (1:10,000, Abcam cat# ab150131, RRID: AB_2732857).
5
Quantitative Analysis of Autophagy Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were grown on coverslips, and at indicated time points after PDT, the cells were washed with PBS for 2 min, fixed with 4% PFA for 20 min, and gently rinsed in 0.3% Triton X-100 for 15 min at room temperature. After washing with PBS for 3 min, the cells were blocked with 5% BSA for 1 h. The cells were then incubated with anti-Lamp2 and anti-LC3-II antibodies (dilution 1:500) overnight at 4°C and washed with PBS for 5 min. Afterward, Alexa Fluor 488-labeled (AB150073; Abcam) and Alexa Fluor 647-labeled (AB150131; Abcam) secondary antibodies (dilution 1:200) were added to develop the fluorescence staining at room temperature. The fluorescence intensity and the co-localization between Lamp2 and LC3-II were observed under a fluorescence microscope (BX41; Olympus Corporation, Tokyo, Japan) and quantitatively analyzed by Image-Pro Plus 6.0.
6
Immunofluorescent Analysis of Kidney Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunofluorescent analysis, kidneys were harvested and fixed in 4% formaldehyde overnight at 4°C, and then embedded in OCT (optimal cutting temperature compound) to obtain frozen-sections at 100 μm on Micron HM550 cryostat. The primary antibodies used were anti-Cox2 (100034-lea, Cayman), anti-Pax2a (ab229318, Abcam), anti-β-catenin (C7207, Sigma), anti-p-Ser9-GSK3 beta (ab107166, Abcam), anti-Pan-cadherin (C3678, Sigma), and anti-phospho-β-catenin (ser675) (D2F1) XP Rabbit mab (4176T, CST). The secondary antibodies used goat anti-mouse IgG H&L Alexa Fluor 647 (ab150115, Abcam), donkey anti-goat IgG Alexa Fluor 647 (ab150131, Abcam), goat anti-rabbit IgG (H+L) Alexa Fluor 633 (A11008, Invitrogen), and goat anti-rabbit IgG (H+L) Alexa Fluor 488 (A21070, Invitrogen), at 1:500. Images were taken using the Nikon A1 confocal microscope. Fluorescent intensities per unit area were measured using ImageJ.
7
Immunofluorescence Staining of Paraffin-Embedded Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The slides of cells were embedded in paraffin according to standard pathology protocols. After antigen retrieval and permeabilization with 0.3% Triton X‐100, tissues were incubated incubated at 4°C overnight with the recommended concentrations of primary Abs (mouse anti‐human antibodies, including C5a (ab11877, Abcam), CCL2 (MABN712, Millipore), and rabbit anti‐human antibodies, such as C5aR1 (ab59390, Abcam), CCR2(NBP1‐48337, Novusbio)) by the manufacturer. After washing, slides were incubated with selected secondary Abs (AF594‐ goat anti‐mouse IgG (ab150116, Abcam), AF488‐ goat anti‐rabbit IgG (ab150077, Abcam), AF647‐ donkey anti‐goat IgG (ab150131, Abcam)) for 40 min at room temperature in the dark, correctly matched to the appropriate species. DAPI mounting medium (Vector Laboratories) was used for cell nuclei staining. Finally, slides were viewed under an imaging fluorescence microscope (Olympus BX51; Olympus).
8
Immunofluorescence Assay for AdipoR1 and AdipoR2 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
OCCM-30 cells were cultured overnight on sterile Falcon™ Chambered Cell Culture Slides (354108, Fisher Scientific) and further fixed with 4% paraformaldehyde (30525-89-4, Sigma-Aldrich) for 10 min at room temperature. Cells were permeabilized with 0.5% Triton™ X-100 Surfact-Amps™ Detergent Solution (28313, Thermo-Fisher) for 20 min. Then, cells were incubated in blocking buffer containing 10% goat serum, 0.3 M glycine, 1% BSA (071M8410, Sigma) and 0.1% Tween-20 (P1379, Sigma-Aldrich) for 30 min at room temperature and further incubated with primary antibodies AdipoR1 (ab70362, Abcam) (dilution 1:250) or AdipoR2 (ab77612, Abcam) (dilution 1:250) at 4°C overnight. The secondary antibodies DyLight 488 polyclonal goat anti-rabbit (ab96899, Abcam) (dilution 1:500) or donkey anti-goat Alexa Fluor 647 (ab150131, Abcam) (dilution 1:500) conjugated to fluorescein isothiocyanate were used. After washing with 1× phosphate-buffered saline (PBS) (10010023, Thermo-Fisher), samples were mounted using a fluorescent Mounting Medium with DAPI (ab104139, Abcam). Staining was analyzed using a high-resolution fluorescence microscope (Leica Microsystems, Wetzlar, Germany) and photographed.
9
Immunofluorescence Staining of Embryonic Stem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ESCs were fixed for 10 min with 4% paraformaldehyde (PFA) at room temperature (RT) permeabilized for 10 min with 0.4% Triton X-100-PBS and blocked for 30 min with 10% goat serum in PBST (PBS with 0.05% Tween 20). Incubation with primary antibodies in blocking buffer was performed overnight at 4 °C. After washing with PBST, cells were incubated in blocking buffer with the secondary antibodies for 1 h at RT. Slides were then washed in PBST and mounted with ProLong® Gold Antifade Mountant with DAPI (Thermo Fisher Scientific). Images were acquired using a fluorescence microscope (Axioplan2; Carl Zeiss) or a confocal Zeiss LSM700 microscope (Carl Zeiss, Jena) with Zen image acquisition software. Images were processed with Fiji and CC Photoshop software (Adobe). The following primary antibodies were used: goat anti-REX1 (Santa Cruz, sc-50670, 1:50), mouse anti-OCT4 (Santa Cruz, sc-5279, 1:100), rabbit anti-RNF12 (a generous gift from Dr. Ingolf Bach, 1:100), rabbit anti-NANOG (Calbiochem, SC1000, 1:100) and rabbit anti-H3K27me3 (Diagenode, C15310069, 1:500). The following Alexa Fluor secondary antibodies were used: donkey anti-mouse 488 (Thermo Fisher Scientific, A-21202, 1:500), donkey anti-rabbit 488 (Thermo Fisher Scientific, A-21206, 1:500), donkey anti-rabbit 546 (Thermo Fisher Scientific, A10040, 1:500) and donkey anti-goat 647 (Abcam, ab150131, 1:500).
10
Visualizing Neutrophil Extracellular Traps
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The lung tissue sections (5 μm) were paraffin-embedded and fixed on the slides. Following deparaffinizing, clearing, and blocking, the sections were incubated with rabbit antibody to citrullinated histone H3 (Cit-H3; 1: 100, ab219407, Abcam, UK) and goat antibody to NE (sc-9520, Santa Cruz Biotechnology, Inc, Santa Cruz, CA, USA) respectively, at 4 °C overnight. The following day, the sections were incubated with secondary antibody, Alexa Fluor 488 goat anti-rabbit antibody to immunoglobulin G (IgG) (1: 200, ab150073, Abcam, UK) and Alexa Fluor 647 goat anti-rabbit antibody to IgG (1: 500, ab150131, Abcam, UK) at room temperature for 1 h. The DNA was stained by 4′,6-diamidino-2-phenylindole (DAPI, Life Technologies, Carlsbad, CA, USA) and the formation of NET was observed under a confocal microscope.
The PMA-treated PMNs were settled in the polylysine-coated slides (Corning Incorporated, Corning, NY, USA) and fixed with 4% polyformaldehyde (Sigma-Aldrich Chemical Company, St Louis, MO, USA) for 30 min. The PMNs were incubated with SYTOX Green (Thermo Fisher Scientific, Waltham, MA, USA) and Hoechst 33,342 (ImmunoChemistry Technologies, Bloomington, MN, USA) for 15 min. After that, the sections were mounted using anti-fluorescence quencher, and NET formation was observed under the confocal microscope.
The PMA-treated PMNs were settled in the polylysine-coated slides (Corning Incorporated, Corning, NY, USA) and fixed with 4% polyformaldehyde (Sigma-Aldrich Chemical Company, St Louis, MO, USA) for 30 min. The PMNs were incubated with SYTOX Green (Thermo Fisher Scientific, Waltham, MA, USA) and Hoechst 33,342 (ImmunoChemistry Technologies, Bloomington, MN, USA) for 15 min. After that, the sections were mounted using anti-fluorescence quencher, and NET formation was observed under the confocal microscope.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!