In order to detect CD39 and CD73 co-expression by immunocytochemistry, fixed synovial cells were stained for these cell surface ectonucleotidases using the primary antibodies rabbit anti-CD39 (ab178572, Abcam, Cambridge, UK) and mouse anti-CD73 (ab133582, Abcam, Cambridge, UK). The expression of ADA, ENT1, ENT2, and ARs was investigated by immunohistochemical staining of synovial tissue samples using the primary antibodies rabbit anti-ADA (NBP1-90361, Novus Biologicals, Cambridge, UK), rabbit anti-ENT1 (NBP1-84838, Novus Biologicals, Cambridge, UK), rabbit anti-ENT2 (ab48595, Abcam, Cambridge, UK), rabbit anti-A1AR (ab124780, Abcam, Cambridge, UK), rabbit anti-A2AAR (ab260032, Abcam, Cambridge, UK), rabbit anti-A2BAR (LS-C20310, LSBio, via Biozol, Eching, Germany), and rabbit anti-A3AR (LS-A686, LSBio, via Biozol, Eching, Germany). After blocking (10% bovine serum albumine, 10% chicken serum, and 10% goat serum), cytospin or tissue slides were incubated with primary antibodies overnight at 4 °C. Primary staining was visualized using Alexa Fluor-labeled secondary antibodies (goat anti-rabbit Alexa Fluor 594 or goat anti-mouse or anti-rabbit Alexa Fluor 488; Thermo Fisher/Life technologies, Schwerte, Germany). Cell nuclei were counterstained with DAPI (Merck, Darmstadt, Germany). Slides without primary antibody served as negative controls (Supplementary Materials Figure S2 ).
Ab260032
Manufactured by Abcam
Sourced in United Kingdom
Ab260032 is a lab equipment product offered by Abcam. It is designed for use in various research applications. The core function of this product is to provide a specific capability or feature required for laboratory work.
Automatically generated - may contain errors
Lab products found in correlation
Ab133582, by Abcam (1 mentions)
Ab124780, by Abcam (1 mentions)
Ab178572, by Abcam (1 mentions)
Ab48595, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Envision flex kit, by Agilent Technologies (1 mentions)
Autostainer link 48 machine, by Agilent Technologies (1 mentions)
Ab32063, by Abcam (1 mentions)
3 protocols using ab260032
1
Immunocytochemistry and Immunohistochemistry of Synovial Adenosine Pathway
2
Immunohistochemistry for ADORA2A and TNFRSF18
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Rabbit monoclonal antibodies for ADORA2A (ab260032) and TNFRSF18 (ab223841) were purchased from Abcam (USA). After slicing into 4-µm sections, all tissues were deparaffinized and treated with ethylenediamine tetraacetic acid (EDTA) (pH 9.0) for antigen retrieval in a microwave for 20 min. We used an Autostainer Link 48 machine (Dako, Denmark A/S, Denmark) for the staining. Subsequently, primary antibodies for ADORA2A (rabbit monoclonal, 1:100 dilution) and TNFRSF18 (rabbit monoclonal, 1:200 dilution) were added to the sections, while phosphate buffered saline (PBS) buffer was used as a blank control instead of the antibody. An EnVision Flex Kit (Dako, Denmark A/S, Denmark) was used as the second antibody. Two senior pathologists examined all cases to validate the initial scores. The percentage of positively stained cells and staining scores were used to assess the IHC results according to the methodology described in previous articles (7 (link),8 (link)). The Gene-Score = (percentage of cells of weak intensity ×1) + (percentage of cells of moderate intensity ×2) + percentage of cells of strong intensity ×3).
3
Immunohistochemical Assessment of Rat Brain and Ovary
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Paraffin blocks of the rat cerebral cortex and ovaries were sectioned and mounted on positively charged slides for immunostaining using rabbit monoclonal antibodies against E2R-α (ab32063, Abcam PLC, Cambridge, UK), A2AR (ab260032, Abcam), and MT2R (ab167108, Abcam). Sections were incubated overnight with two drops of the primary antibody, followed by incubation for 10 min with two drops of biotinylated goat anti-polyvalent secondary antibody, and finally with two drops of streptavidin-peroxidase for an additional 10 min. Secondary antibodies were produced by immunizing goat with rabbit IgG, yielding goat anti-rabbit IgG secondary antibodies. Diaminobenzidine (DAB) was used as a chromogen, while Meyer's hematoxylin was used as a counterstain. The positive cortical and ovarian controls for E2Rα, A2AR, and MT2R were pituitary tissues with membranous and cytoplasmic reactions, and ovarian carcinoma sections with brownish cytoplasmic, nuclear, and membranous reactions, respectively.
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