Cancer cells‐T cells coculture system consisted of MKN‐45 and human T cells in the ratio of 10:1; the cells were planted into a 4‐well‐Millicell® EZ SLIDES (Millipore) with 10% Matrigel (Corning, Corning, NY, USA). The sides were stained for co‐staining of in situ hybridization (ISH) and immunocytochemistry (ICC) staining according to a previous publication.24 miR‐451 was detected with a Cy3 labeled probe to miR‐451 (HM451‐100) (BioGenex, Fremont, CA, USA), T cells were detected with antibody targeting CD3 (ab699; Abcam, Cambridge, MA, USA) followed by goat anti‐mouse IgG H&L (Alexa Fluor® 647) (ab150115; Abcam) and exosome was detected by using antibody targeting CD63 (ab68418; Abcam) followed by goat anti‐rabbit Cy5 labeled second antibody (ab97077; Abcam). Slides were finally mounted with Fluoroshield Mounting Medium With DAPI (ab104139; Abcam).
Ab699
Manufactured by Abcam
Sourced in United States
Ab699 is a laboratory equipment product offered by Abcam. It is a device used for the purpose of scientific research and experimentation. The core function of Ab699 is to perform a specific task or measurement related to the needs of the laboratory environment.
Automatically generated - may contain errors
Lab products found in correlation
Ab134114, by Abcam (2 mentions)
Ab133616, by Abcam (2 mentions)
Ab68418, by Abcam (1 mentions)
Ab97077, by Abcam (1 mentions)
Millicell ez slide, by Merck Group (1 mentions)
Ab104139, by Abcam (1 mentions)
Matrigel, by Corning (1 mentions)
Ab150115, by Abcam (1 mentions)
Ab5978, by Abcam (1 mentions)
Ab5980, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Ab53013, by Abcam (1 mentions)
Ab124824, by Abcam (1 mentions)
Ab75985, by Abcam (1 mentions)
Ab78237, by Abcam (1 mentions)
Ab6672, by Abcam (1 mentions)
Ab137132, by Abcam (1 mentions)
Ab2406, by Abcam (1 mentions)
Ab190958, by Abcam (1 mentions)
12 protocols using ab699
1
Cancer-T Cell Coculture and Imaging
2
Immunofluorescence Staining of Aortic Valve
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Cells in the samples were collected and washed twice in PBS before being fixed in 2 mL 4% paraformaldehyde for 10min.After that,the samples were washed twice in PBS and permeabilized by incubation with 2 mL of 0.1% Triton X-100 in PBS for 15 min. Then, after washing thrice with PBS and blocking for 1 h, Cells were incubated with IL-21R (ab5980, diluted 1:100), antibody overnight and washed for three times. Subsequently, they were incubated with second antibody in a dark humidity chamber at 4 °C for 1 hand subsequently washed five times with PBS. For immunofluorescence staining of human normal and calcified aortic valve tissue, slices were incubated with anti-IL-21(ab5978, 1:100), anti-IL-21R (ab5980, diluted 1:100), antiCD3 (Abcam, ab699, 1:1000), anti-IL-a-SMA (ab119542, 1:100), overnight, subsequently, they were incubated with second antibody in a dark humidity chamber at 4 °C for 1 hand subsequently washed five times with PBS. Nuclei were stained with DAPI (solarbio, C0060l, diluted 1:5000). Cells and tissue samples were viewed by a fluorescence microscope (Olympus).
3
Immunohistochemical Assessment of CXCR4, CD Markers, and TLR9 in LSG Biopsies
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Immunohistochemical staining to detect C-X-C chemokine receptor type 4 (CXCR4), CD19, CD21, Toll-like receptor 9 (TLR9) and intercellular cell adhesion molecule 1 (ICAM1) was performed on LSG biopsy sections as described previously [25 (link)]. CXCR4 (ab124824, Abcam, Cambridge, MA, USA), CD3 (ab699, Abcam, Cambridge, MA, USA), CD19 (ab134114, Abcam, Cambridge, MA, USA), CD21 (ab75985, Abcam Cambridge, MA, USA), ICAM1 (ab53013, Abcam, Cambridge, MA, USA), CD20 (ab78237, Abcam, Cambridge, MA, USA), and TLR9 (BA3861-1, Boster Biotechnology, Wuhan, China) antibodies were used in this experiment. Negative control staining was performed by replacing primary antibodies with PBS. Positive immunoreactivity appeared as a brown color. Double staining for CD3 and CD20 was used to analyze T/B cell segregation using the DouMaxvision™ double-stain system (KIT-9998, Maixin Biotechnology, Fuzhou, China). A scoring system was used to describe the results of double staining as described previously [26 (link)]. The images of grade 1 to 3 are shown in Additional file 2 : Figure S3.
4
Histopathological Analysis of Mouse Livers
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Mouse livers were collected at the specified time points, fixed with 10% neutral buffered formalin and embedded in paraffin for processing into 4 micron sections. Sections were rehydrated and stained with Haematoxylin and Eosin (H&E) (Thermo Scientific) or Sirius red and Fast green (Sigma) before pathology evaluation. Alternatively, sections were treated with heat mediated antigen retrieval with pH6 sodium citrate or pH9 Tris-EDTA buffer before staining with primary antibodies. Primary antibodies used in this study include anti-HSA (ab2406, ab19180 Abcam), anti-CD3 (ab699, Abcam), anti-CD20 (555677, BD), anti-CD68 (ab955, Abcam), anti-PD1 (ab137132, Abcam), anti-β-catenin (610154, BD), anti-glutamine synthetase (MAB302, Millipore), anti-LFABP (ab190958, Abcam), anti-IL6 (ab6672, Abcam) and anti-IL10 (ab34843, Abcam).
Immunohistochemistry staining was performed using the Superpicture 3rd Gen IHC Detection Kit (879673, Life Technologies) while immunofluorescence staining was done using anti-mouse IgG, anti-rabbit IgG or anti-Goat IgG secondary antibodies conjugated to Alexafluors 488 or 647 (Life Technologies). Images were captured using an Olympus upright confocal microscope or a Zeiss Axioscan Z1 slide scanner.
Immunohistochemistry staining was performed using the Superpicture 3rd Gen IHC Detection Kit (879673, Life Technologies) while immunofluorescence staining was done using anti-mouse IgG, anti-rabbit IgG or anti-Goat IgG secondary antibodies conjugated to Alexafluors 488 or 647 (Life Technologies). Images were captured using an Olympus upright confocal microscope or a Zeiss Axioscan Z1 slide scanner.
5
Immunohistochemical Analysis of Immune Markers
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CD3 (ab699, Abcam), CD4 (ab133616, Abcam), CD8 (ab93278, Abcam), and PD-L1 ([SP142]-C-terminal, prediluted, Abcam) were used to test expression of the corresponding proteins. Tissue sections 5-μm-thick were deparaffinized and dehydrated. Endogenous peroxidase was blocked with 3% hydrogen peroxide for 10 min at room temperature. Antigen retrieval was performed in Ethylene Diamine Tetraacetic Acid (EDTA), for 2 min at 100 °C. The slides were incubated with the primary antibody at 37 °C for 2 h. After three washes with phosphate-buffered saline, the slides were co-incubated with horseradish peroxidase-labelled goat anti rabbit/mouse secondary antibodies. The slides were counter-stained with hematoxylin. Each slide was examined by an experienced pathologist to obtain the coincident immunohistochemical results.
6
Immunostaining of Patient Tissues
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Bake slides of patient tissues were washed with PBS, permeabilised with 0.3% Triton X‐100 in PBS, and then blocked in 5% BSA followed by overnight incubation with primary antibodies at 4°C. The primary antibodies used for immunostaining were human anti‐CD3 (ab699, Abcam, UK), human anti‐CD19 (ab134114, Abcam, UK), and purified anti‐human TCR Vα7.2 (351702, Biolegend, USA). After washing, sections were incubated with secondary antibodies at room temperature for 2 h. Images were taken by an Olympus fluorescence microscope (BX61, OLYMPUS, Japan) and a confocal microscope (Lecia TCS SP8).
7
Grading Inflammatory Cell Infiltration in CNS
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Brains and spinal cords were fixed in 4% buffered formalin fixative overnight. Paraffin wax embedded sections (5 µm) were stained with hematoxylin and eosin and CD3 (ab699; Abcam) immunohistochemical staining. The slides were analyzed on light microscope (BX51; Olympus), and digital images were acquired by digital camera. The level of infiltration was graded using the following score: 0, no inflammatory cells; 1, a few scattered inflammatory cells; 2, organization of inflammatory infiltrates into perivascular cuffs; 3, extensive perivascular cuffing with extension into adjacent subarachnoid space and CNS parenchyma, and 4, extensive perivascular cuffing with increasing subarachnoid and parenchymal inflammation (32 (link)). Slides were analyzed on Olympus BX51 microscope, and digital images were acquired by Olympus digital camera (DP71).
8
Quantifying TWEAK and CD3 in IBD
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Immunofluorescence was performed to detect TWEAK and CD3 in colon tissues of patients with CD, UC, and normal patients (n = 10/group) with anti-TWEAK (Ab73170; Abcam) at a concentration of 20 μg/mL and anti-CD3 antibody (Ab699; Abcam) 1/40 dilution. An isotype antibody was used as a negative control.
9
Multiplex Immunofluorescence Analysis of Tumor-Infiltrating Immune Cells
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Multiplex immunofluorescence staining with a four-color IHC kit (Absin, China) was performed on tumour samples from CBR group and NCB group patients for counting immune cells infiltrating different tumours. The slides were deparaffinized in xylene, rehydrated, and washed in ethylenediaminetetraacetic acid (EDTA)-sodium citrate buffer for microwave antigen retrieval. Endogenous peroxidase activity was blocked using an antibody diluent/blocking agent (PerkinElmer, USA). Primary antibodies CD3 (1:500, Abcam, ab699), CD8 (1:500, Abcam, ab101500), and CD68 (1:500, Cell Signalling Technology, E3O7V) were incubated at room temperature for 1 h. Polymer HRP Ms + Rb was incubated for 10 min at 37 °C. Subsequently, the slides were incubated at room temperature for 10 min with TSA fluorescent dyes (TSA520, TSA570, and TSA650) diluted in signal amplification solution. Antigen-antibody complexes were stripped by microwave treatment using 0.05% Tris–EDTA buffer. TSA single-stained slides were counterstained with DAPI for 5 min and then coverslipped. Three observers, blinded to the experimental design of each sample, evaluated random fields of view at 100x magnification and calculated the number of immune cells in each field. Images were acquired using a confocal laser-scanning microscope (Zeiss Microscopy, USA).
10
Immunofluorescence Staining of Skin Samples
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For immunofluorescence (IF) analysis, the samples were harvested from the skin of the hind limb of interest at a size of 2 × 2 cm. Samples were then fixed with 10% formaldehyde for 12 to 14 days at room temperature, embedded in paraffin and sectioned for H&E and IF staining. The slides were rehydrated, and heat‐induced antigen retrieval was performed using 95°C citric acid (S2369, Dako). Nonspecific binding was blocked with antibody diluent (S3022, Dako) for 30 min. Samples were incubated with the following primary antibodies overnight at 4°C: anti‐LYVE‐1 (1:200, ab10278, Abcam), anti‐collagen I (1:200, ab34710, Abcam), anti‐CD3 (1:200, ab699, Abcam), anti‐CD4 (1:200, ab133616, Abcam), anti‐CD45 (1:200, ab10558, Abcam), and anti‐alpha smooth muscle actin (α‐SMA) (1:200, ab21027, Abcam) and anti‐podoplanin (1:200, 14–5381‐85, Invitrogen). IF staining was performed using the following secondary antibodies: Alexa Fluor® 488‐conjugated donkey anti‐rabbit IgG (1:500, ab150061, Abcam), Alexa Fluor® 488‐conjugated goat anti‐mouse IgG (1:500, ab150117, Abcam), Alexa Fluor® 594‐conjugated goat anti‐mouse IgG (1:500, ab150120, Abcam) and Alexa Fluor® 594‐conjugated goat anti‐rabbit IgG (1:500, ab150080, Abcam). Nuclear staining was performed using DAPI.
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