Autoclaved 22x22 mm cover glasses (3506; Thermo Fisher Scientific) were placed into wells prior to cell seeding and cytokine treatments that were previously described. Following treatment, cells on cover glasses were fixed in 4% paraformaldehyde for 15 minutes and thoroughly washed with DPBS. Cells were permeabilized with ice-cold 100% methanol for 10 minutes at −20°C. Cells were blocked for endogenous peroxidase activity with Bloxall (SP-6000; Vector Labs) for 10 minutes. Cells were then blocked with 5% goat serum (Sigma) for 1 hour and incubated in anti-Phospho-SMAD2/3 (1:100; D27F4; Cell Signaling) primary antibody overnight at 4°C. Cells were then incubated in biotinylated goat-anti-rabbit secondary (1:500; B2770; Invitrogen) for 45 minutes at 37°C and subsequently exposed to ready-to-use peroxidase (PK-7100; Vector Labs). Color was developed using Nova Red substrate (SK-4800; Vector Labs). Micrographs were captured with a Nikon Labophot-2 microscope with Nuance software version 3.0.0.
B 2770
Manufactured by Thermo Fisher Scientific
Sourced in United States
The B-2770 is a laboratory centrifuge designed for general purpose applications. It features a maximum speed of 4,000 RPM and a maximum centrifugal force of 2,770 RCF. The centrifuge accommodates a range of rotor and sample tube sizes to support various sample processing needs.
Automatically generated - may contain errors
Lab products found in correlation
Labophot 2 microscope, by Nikon (1 mentions)
Bloxall sp 6000, by Vector Laboratories (1 mentions)
Pk 7100, by Vector Laboratories (1 mentions)
Novared substrate, by Vector Laboratories (1 mentions)
A11008, by Thermo Fisher Scientific (1 mentions)
A11012, by Thermo Fisher Scientific (1 mentions)
A11001, by Thermo Fisher Scientific (1 mentions)
A11005, by Thermo Fisher Scientific (1 mentions)
G3893, by Thermo Fisher Scientific (1 mentions)
Mn1020, by Abcam (1 mentions)
Pa5 16291, by Thermo Fisher Scientific (1 mentions)
Colorview 2 camera, by Olympus (1 mentions)
Ix71 microscope, by Olympus (1 mentions)
Abc reagent, by Vector Laboratories (1 mentions)
Normal horse serum blocking solution, by Vector Laboratories (1 mentions)
Dab peroxidase substrate, by Vector Laboratories (1 mentions)
Ab15191, by Abcam (1 mentions)
Bs 0782r, by Bioss Antibodies (1 mentions)
Pk 6100, by Vector Laboratories (1 mentions)
4 protocols using b 2770
1
Immunostaining for Phospho-SMAD2/3
2
Histological Analysis of Alzheimer's Markers
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Samples were fixed and cut to 50 μm thick for Nissl staining and immunolabeling. The slices were incubated in primary antibody solution containing mouse anti‐Aβ (Invitrogen MA1‐91209; 1:500), mouse anti‐phospho‐tau Ser202‐Thr205 (Invitrogen MN1020; 1:500), rabbit anti‐vimentin (Abcam ab137321; 1:500), mouse anti‐GFAP (Sigma‐Aldrich G3893; 1:500), rabbit anti‐GFAP (Invitrogen PA5‐16291; 1:500), rabbit anti‐GS (Invitrogen 701989; 1:500), rabbit anti‐GLAST (Invitrogen PA5‐19709; 1:500), rabbit anti‐IBA1 (Wako 019‐19741; 1:500), rabbit anti‐AQP4 (Sigma‐Aldrich HPA014784; 1:500), or mouse anti‐claudin‐5 (Invitrogen 35‐2500; 1:500). Subsequently, the samples were incubated with corresponding secondary antibody solution containing biotinylated goat anti‐mouse (Invitrogen 31800; 1:250) or goat anti‐rabbit (Invitrogen B‐2770; 1:250) for immunohistochemistry, and goat anti‐mouse Alexa Fluor 488 (Invitrogen A‐11001; 1:750), goat anti‐mouse Alexa Fluor 594 (Invitrogen A‐11005; 1:750), goat anti‐rabbit Alexa Fluor 488 (Invitrogen A‐11008; 1:750), or goat anti‐rabbit Alexa Fluor 594 (Invitrogen A‐11012; 1:750) for immunofluorescence. The latter also contained human endothelial marker DyLight 649 lectin UEA I (Vector Labs DL‐1068; 1:500).
3
Quantifying Aortic Oxidative Stress Markers
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Aortic ring segments (3–4 mm) with intact adventitia and perivascular fat were fixed in paraformaldehyde (4%), paraffin-embedded and cut into sections of 5 µm. After deparaffinization, samples were blocked with 2.5 % normal horse serum blocking solution (Vector) and stained with primary antibodies against either NADPH-oxidase 2 (Nox-2, 1:200, LSBio, LS-B12365, Seattle, WA, USA) or 3-NT (1:200, Merck Millipore, 06-284:) and biotinylated with a secondary antibody (1:1000, B-2770, Invitrogen, Carlsbad, CA, USA). The ABC reagent (Vector) with DAB peroxidase substrate (Vector) was used for immunohistochemical detection. The brown precipitate formed by oxidized DAB was visualized using an Olympus IX71 microscope (40× objective) and an Olympus Color View II camera (Shinjuku, Tokyo, Japan). Image J software (NIH, USA) was used to measure the level of expression of Nox2 and 3-NT positive proteins in aortic tissue. For accuracy, measurements were performed twice and averaged by two blinded investigators, values were expressed as the % of stained aortic area.
4
Immunohistochemical Analysis of IL-6 and MMP3
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Following fixation in formalin for 48 h, AF samples were washed under running tap water for 2 h, dehydrated and embedded in paraffin-wax. Crosssections with 7 µm thickness were dewaxed and rehydrated. For antigen retrieval, the sections were incubated with 10 mmol/L citrate buffer (pH 6.0, 85 °C, 20 min), followed by hyaluronidase (2 mg/ mL in citrate buffer, pH 8.0, 30 min, 37 °C) and collagenase (2 mg/mL in citrate buffer, pH 8.0, 15 min, 37 °C) digestion. The avidin-biotin complex kit (PK-6100, Vector laboratories) and Vector ® NovaRED ® Substrate Kit, Peroxidase (HRP) (SK-4800, Vector Laboratories) were used for the immunostaining. Sections were incubated with rabbit anti-IL-6 (1 : 200, bs0782R, Bioss, Woburn, MA, USA) or rabbit anti-MMP3 (1 : 200, ab15191, Abcam) antibodies overnight at 4 °C, according to Saggese et al. (2019) . Goat antirabbit IgG Biotin-XX (1 : 200, B-2770, Invitrogen) was used as secondary antibody. Primary antibodies were polyclonal and reacted with human and bovine molecules. All samples from the same experiment were stained at the same time for each marker for comparison purposes.
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