Immediately following OGD 6 h/reoxygenation 6 or 24 h with vehicle or ATN-161 treatment, the cells were fixed in 100% methanol, washed with PBS, and then incubated with peroxiabolish (Biocare Medical, Concord, CA) for 10 min to block endogenous peroxidase. The cells were washed with PBS, blocked with Rodent M Block (Biocare Medical, Concord, CA) for 15 min at room temperature, and then incubated with primary antibodies (Supplementary Table 2 ) prepared in DaVinci diluent (Biocare Medical, Concord, CA) overnight at 4 °C. The cells were then washed with PBS four times and incubated with Alexa Fluor 568 or 488 conjugated secondary antibodies (Supplementary Table 2 ) (Life Technologies; 1:400), for 45 min at room temperature. After three washes with PBS, cells were mounted using Vectashield containing DAPI (Vector Labs, Burlingame, CA, USA). The images were obtained using a fluorescence microscope (TS2R, Nikon, Tokyo, Japan). The antibody fluorescence positive pixels per cell was quantified using ImageJ analysis software (version 1.41; NIH).
Da vinci diluent
Manufactured by Biocare Medical
The Da Vinci diluent is a laboratory reagent used to dilute samples prior to analysis. It is a sterile, isotonic solution that helps maintain the integrity of the samples during the dilution process.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 568 or 488 conjugated secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Rodent block m, by Biocare Medical (1 mentions)
Vectashield containing dapi, by Vector Laboratories (1 mentions)
Rabbit anti mouse cd45, by Cell Signaling Technology (1 mentions)
Pt link, by Agilent Technologies (1 mentions)
Pannoramic midi 2 scanner, by 3DHISTECH (1 mentions)
Tacha s hematoxylin, by Biocare Medical (1 mentions)
Mach2 rabbit ap polymer, by Biocare Medical (1 mentions)
Dako autostainer link 48 platform, by Agilent Technologies (1 mentions)
Dako coverstainer, by Agilent Technologies (1 mentions)
Halo software, by Indica Labs (1 mentions)
Decloaking chamber, by Biocare Medical (1 mentions)
3 protocols using da vinci diluent
1
Immunofluorescence Staining for Cellular Analysis
2
Immunohistochemical Profiling of Murine Tissues
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Tissues were fixed in 10% neutral buffered formalin, processed and embedded in paraffin, and sectioned at 4 μm thickness using standard histologic procedures. Slides were dewaxed using xylene and rehydrated with a graded series of ethanol using a DAKO Coverstainer (DAKO, Agilent Technologies). Antigen retrieval was performed in a high pH buffer using PT Link (DAKO, Agilent Technologies) at 95°C for 20 minutes. IHC was carried out using a DAKO Autostainer Link 48 platform (DAKO, Agilent Technologies). Briefly, slides were blocked for endogenous peroxidases and subsequently stained using the following primary antibodies: rabbit anti-mouse CD8 (D4W27 at 1:200), Rabbit anti-mouse CD45 (Cell Signaling Technology D3F8Q at 1:200), and Rabbit anti-mouse CD31 antibodies (Abcam EPR17259 at 1:500) in Da Vinci diluent (Biocare Medical). MACH2 Rabbit AP polymer (Biocare Medical) was used to detect primary antibodies, followed by detection using Enzo Red chromogen (Enzo Life Sciences). Slides were then counterstained with Tacha's Hematoxylin (Biocare Medical), dehydrated, and coverslipped using the DAKO Coverstainer. Once dried, slides were scanned using a 3D Histech Pannoramic MIDI II Scanner (3DHISTECH), then image/spatial analyses were performed using HALO software (Indica Labs).
3
Quantitative CD31 Immunohistochemistry Assay
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Immunohistochemistry for CD31 was applied to each of the above-mentioned specimens. The monoclonal anti-CD31 antibody (clone JC/70A, Dako-Agilent, Carpinteria, CA) recognizes a fixation-resistant epitope in endothelial cells. 31 The slides were immersed in Reveal (Biocare Medical, Concord, CA) antigen retrieval solution in a decloaking chamber (Biocare) for 30 seconds at 125 C and 18 to 24 PSI. The primary antibody was diluted at 1/100 using Da Vinci diluent (Biocare Medical) and incubated for 60 minutes. An immunoperoxidase, polymer-based detection system was used (PromARK Mouseon-Canine HRP-Polymer, Biocare Medical). The chromogen was diaminobenzidine (DAB). All incubations were at room temperature. Randomly selected sections of test samples were treated with Biocare Medical polymer negative control serum in place of the primary antibody. The target cell population was considered positive if at least 10% of the cells had a distinct membranous labeling for CD31. The number of positive cells was scored semiquantitatively in the hepatic and renal lesions and plasmacytomas as follows: score 1 (10%-20% positive cells), 2 (21%-40%), 3 (41%-60%), and 4 (>60%). The intensity of the reaction was subjectively graded as weak, moderate, or strong based on the target area showing the highest intensity in each sample.
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