Aortas from euthanized mice were isolated after perfusion with saline followed by 4% paraformaldehyde. Aortas were then longitudinally cut and pinned to a gelatin-coated plate and fixed in 4% paraformaldehyde overnight at 4 °C. The tissue was permeabilized and blocked with 0.3% Triton X-100, 10% goat serum, 5% BSA, and FcBlock (1:100; Rat Anti-Mouse CD16/CD32; BD Bioscience) in PBS for 1 h. Aortas were incubated overnight at room temperature hamster anti-cd31 (1:200; MAB1398Z; Millipore) in blocking buffer. After abundant washes with PBS, samples were incubated overnight with AlexaFluor647-conjugated goat anti-hamster secondary antibody (1:500, 127-605-160, Jackson Inmuno Reasearch) and DAPI (1:10,000). Aortas were mounted in Citifluor AF4 mounting medium (Aname). Images were acquired with a Nikon A1R confocal microscope fitted with a 20x air objective or a 40x oil immersion objective and using Nikon NIS-Elements software (1024 × 1024 pixels, 8bits).
Mab1398z
Manufactured by Merck Group
Sourced in United States
MAB1398Z is a laboratory equipment product from Merck Group. It is designed for general laboratory use. The core function of MAB1398Z is to perform specific laboratory tasks, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710, by Zeiss (2 mentions)
Lsm 510 meta confocal microscope, by Zeiss (2 mentions)
Alexa fluor 488, by Jackson ImmunoResearch (2 mentions)
Lsm800 confocal microscope, by Zeiss (2 mentions)
Prolong diamond antifade reagent, by Thermo Fisher Scientific (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Rat anti mouse cd16 cd32, by BD (1 mentions)
Nis elements software, by Nikon (1 mentions)
A1r confocal microscope, by Nikon (1 mentions)
Ab196149, by Abcam (1 mentions)
Ab4761, by Abcam (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Ab9610, by Merck Group (1 mentions)
Ab182136, by Abcam (1 mentions)
D9542, by Merck Group (1 mentions)
Bodipy, by Thermo Fisher Scientific (1 mentions)
Ab2728, by Abcam (1 mentions)
Ab62623, by Abcam (1 mentions)
Ab109367, by Abcam (1 mentions)
3 3 diaminobenzidine substrate, by Vector Laboratories (1 mentions)
19 protocols using mab1398z
1
Aortic Endothelial Cell Immunofluorescence
2
Immunohistochemistry Protocol for Embryonic Tissues
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For immunohistochemical procedures, embryos were fixed in 4% PFA in PBS 0.1M pH 7.2 by immersion or perfusion depending on the developmental stage, cryoprotected in a 30% sucrose solution, and then embedded in OCT and sectioned in the cryostat at 20 μm thickness in the transverse plane. Immunohistochemistry was performed following standard protocols. Primary antibodies used include: polyclonal anti-CD31 hamster (1:1000; MAB1398Z, Millipore), anti-β-galactosidase rabbit (1:1000; ab4761, Abcam), anti-FoxA2 mouse (1:250; F55A10, DSHB), anti-Nkx6.1 mouse (1:1000; 4C7, DSHB), anti-Olig2 rabbit (1:1000; AB9610, Chemicon), anti-ERG-647 rabbit (1:500; ab196149, Abcam) and anti-WT-1 mouse (Wilms´ Tumor-1; 1:50; M3516, Dako). Sections were incubated with the primary antibody diluted in PBS containing 0.1% Triton X-100 and 1% bovine serum albumin (BSA), for 48 h at 4°C. Subsequently, the sections were rinsed in PBS and incubated for 2 hours at room temperature with 488 or 594-Alexa™-conjugated fluorescent antibodies (Molecular Probes, 1:1000). Sections were counterstained with Hoechst (Molecular Probes, 1:1000) for 5 min at room temperature to visualize nuclei.
3
Whole-mount Immunofluorescence Visualization
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Whole-mount immunofluorescence analyses were performed as described with minor modifications [25 (link)]. Whole-mount tissues were incubated with primary antibody against PECAM-1 (1:200 dilution, MAB1398Z; Millipore, Billerica, MA, USA), ADRB1 (1:200 dilution, PA1-049; Thermo Fisher Scientific, Waltham, MA, USA), ADRB2 (1:200 dilution, ab182136; Abcam), and ADRB3 (1:200 dilution) for 60 h at 4°C. After six washes of 20 min each, the tissues were incubated overnight with the species-specific secondary antibody. The stained whole-mount tissues were subsequently incubated with a fructose solution as an optical clearing agent to diminish the amount of light scattering. The adipocytes and nuclei in whole-mount VAT were stained with BODIPY (1:1,000 dilution, D-3835; Invitrogen) and DAPI (1:1,000 dilution, D9542; Sigma-Aldrich). All images were captured under a confocal microscope (Zeiss confocal LSM 700 laser scanning microscope) at a magnification of ×100 or ×500.
4
Visualizing Choroidal Vasculature and ET-1
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Eyeballs were fixed in 1% PFA (paraformaldehyde) for 1 h to fix choroidal vasculature. We washed away the PFA with PBS and produced the RPE/choroidal/scleral whole mounts by separating the conjunctivae, corneas, lenses, and retinas. The mounts were incubated for 2 h at 37°C, and the RPE was washed away with PBS. The mounts were fixed in 1% PFA for 1.5 h again. After washing the PFA, the mounts were blocked using 0.5% Triton X-100/5% BSA overnight and incubated with the primary antibodies of ET-1 (1∶100; ab2728, Abcam, MA, USA) and CD31 (1∶100; MAB1398Z, Millipore, MA, USA) for 2 days at 4°C. The secondary antibodies of anti-mouse IgG antibody and anti-hamster IgG antibody (1∶800; Alexa Fluor® 488 Conjugate and Alexa Fluor® 647 Conjugate, CST, USA) were added for 1 h. The mounts were photographed using a Zeiss confocal microscope (LSM710, Carl Zeiss).
5
Histological Analysis of Tissue Samples
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For histological examination, hematoxylin and eosin (H&E) staining, and Movat pentachrome staining were performed. Anti-CD31 (MAB1398Z; Millipore, Burlington, MA), anti-alpha-smooth muscle actin (anti-α-SMA; A5228; Sigma-Aldrich, St. Louis, MO; and ab5694; Abcam, Cambridge, United Kingdom), anti-CD45 (Mab114; R&D Systems, Minneapolis, MN), anti-PRDX2 (ab109367; Abcam), anti-4-hydroxynonenal (anti-4-HNE; ab46545; Abcam), anti-DNA/RNA damage antibody (anti-8-OHG) (ab62623; Abcam), and anti-mouse macrophage/monocyte antibody (anti-MOMA2) (MCA519G; Bio-Rad, Hercules, CA) were used as primary antibodies for immunostaining. After incubation with the primary antibodies, Alexa 488 and 594 (Invitrogen, Carlsbad, CA) or biotinylated secondary antibodies with 3,3′-diaminobenzidine substrate (Vector Laboratories, Burlingame, CA) were used to visualize the antigens. Furthermore, 4′,6-diamidino-2-phenylindole (DAPI) or hematoxylin was used to label the nuclei. The negative control tissues were prepared in a similar manner using IgG isotype control antibodies (Santa Cruz Biotechnology, Dallas, TX). The immunofluorescence was imaged with an LSM 510 meta confocal microscope (Carl Zeiss, Oberkochen, Germany) or a BX53 microscope (Olympus, Tokyo, Japan).
6
Immunohistochemical Analysis of Tibialis Anterior
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Tibialis anterior cryosections were fixed in 4% paraformaldehyde for 30 min. Slides were then permeabilized in 0.5% Triton X-100 solution and then blocked in 5% BSA solution for 1 h. The slides were then incubated with CD31 antibody (Millipore MAB1398Z) overnight at 4 °C. The slides were then incubated with secondary antibodies and WGA (ThermoFisher W6748) for 4 h and mounted with VECTASHIELD mounting media with DAPI.
7
Immunostaining Protocol for CD31 in Cells
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For immunostaining, cells were fixed in 4% paraformaldehyde in PBS for 10 min at room temperature. After washing three times in PBS – 0.1% BSA for 5 min, cells were permeabilized with 0.2% Triton (Sigma, T9284) in PBS for 20 min, and washed in PBS – 0.1% BSA. Primary antibody against CD31 (MAB1398Z, Millipore) was diluted in PBS – 0.1% BSA to 1/200 and incubated overnight at 4 °C. After washing, cells were incubated with a FITC-conjugated goat anti-hamster secondary antibody (127-095-099, Jackson, Bar Harbor, ME, USA) used at a 1/1000 dilution in the presence of DAPI for 45 min (1 h at room temperature). Vectashield mounting medium without DAPI (Vectors Lab, Burlingame, CA, USA; H-1000) was applied to all slides. Fluorescent images were taken using a Nikon A1-R inverted confocal microscope.
8
Adipose Tissue Lipid and Vasculature Imaging
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Perigonadal white adipose tissues (pWATs) were isolated (about 0.5 cm3) and immersed in 1% PFA for 1 h and then washed by PBS. For LipidTOX imaging, tissues were stained by HCS LipidTOX™ Deep Red Neutral Lipid Stain (1:500, H34477, Thermo Fisher Scientific) for 30 min, images were obtained on an inverted Leica TCS SP8 confocal microscope (Leica, Germany), and 3D images were composited by taking z-stack images using LAS X software-determined levels along the vertical axis. For PECAM-1 imaging, tissues were sealed using 1% goat serum (SL038, Solarbio LIFE SCIENCES, Beijing, China) for 1 h and then incubated with PECAM-1 antibody (1:1,000, MAB1398Z, Millipore, United States) overnight at 4°C. Tissues were washed and incubated with Goat Anti-Armenian hamster IgG H&L (Alexa Fluor® 647) (1:1,000, ab173004, Abcam, United States) for 1 h, images were obtained on an inverted confocal microscope and 3D images were again composited using the LAS X software.
9
Assessing p16INK4a Expression in Mouse Eyes
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Eyes were fixed in 4% paraformaldehyde for 1 h at RT and then dehydrated in 30% sucrose solution overnight. Afterward, dehydrated eyes were embedded in Tissue-Tek® optimum cutting temperature compound (Sakura Finetek, Japan) and cross-sectioned on a cryostat vertically through the center of the cornea and optic nerve. Serial 15-μm-thick frozen eyes sections were cut using a cryostat (NX70, Thermo Fisher Scientific, US). The eye sections were permeabilized with 0.3% Triton X-100/PBS for 30 min at RT, blocked with 2% BSA, and 0.3% Triton X-100 PBS for 1 h at RT, and then incubated at 4°C overnight with anti-CDKN2A/p16INK4a (ab211542, 1:120; Abcam, UK) and anti-CD31 (MAB1398Z, 1:120, Millipore, US) antibodies. After being washed in PBS, the sections were incubated with Alexa Fluor 488 (1:300, Jackson ImmunoResearch, US) and Alexa Fluor 594 (1:300, Jackson ImmunoResearch, US) conjugated secondary antibodies for 2 hours and 4,6-diamidino-2-phenylindole (DAPI, 1:10,000; Sigma-Aldrich, US) for 5 minutes. Images were taken with a scanning laser confocal microscope (DMI6000B with TCS SP8 system; Leica, Wetzlar, Germany). The p16INK4a positive area was calculated with ImageJ software (ROI tool).
10
Immunocytochemistry of Cellular Markers
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