Paraffin embedded sections of human eyes were deparaffinized and rehydrated with a graded alcohol series. Immunofluorescent staining was performed with antibodies (Abs) against human von Willebrandt factor (F3520, Sigma), human MMR (CD206, MAB2534, R&D Systems), human CD80 (ab53003 Abcam) or human ROCK1 (sc-17794, Santa Cruz Biotechnology) or ROCK2 (sc-1851, Santa Cruz Biotechnology) and identified with Alexa Fluor 488 (10μg/ml, A-11055; Invitrogen) or 647 (10μg/ml, A21244; Invitrogen) secondary Abs. On day 3 after laser injury, 10μm frozen sections of the posterior segment were prepared. The mouse eye sections were incubated with a rat anti-mouse F4/80 mAb (MCA497R, AbD Serotec) or CD11b (550282, BD Pharmingen), and subsequently with the secondary Ab. In monkey eyes, CD68 (goat pAb, sc-7082, Santa Cruz), vWF (rabbit pAb, A0082, DAKO), ROCK1 (mouse mAb, sc-17794, Santa Cruz) and ROCK2 (goat pAb, sc-1851, Santa Cruz) were stained. Images were obtained with a Leica microscope.
Mca497r
Manufactured by Bio-Rad
Sourced in United Kingdom, United States
The MCA497R is a laboratory equipment designed for general research purposes. It serves as a biological tool for analysis and experimentation. The core function of this product is to perform specific tasks related to life science research. Further details on the intended use or capabilities of this equipment are not available.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710, by Zeiss (2 mentions)
A11055, by Thermo Fisher Scientific (1 mentions)
A0082, by Agilent Technologies (1 mentions)
A 21244, by Thermo Fisher Scientific (1 mentions)
F3520, by Merck Group (1 mentions)
Sc 17794, by Santa Cruz Biotechnology (1 mentions)
Sc 1851, by Santa Cruz Biotechnology (1 mentions)
Cd11b, by BD (1 mentions)
Ab61682, by Abcam (1 mentions)
Ab10983, by Abcam (1 mentions)
A32814, by Thermo Fisher Scientific (1 mentions)
A32754, by Thermo Fisher Scientific (1 mentions)
Ab79056, by Abcam (1 mentions)
Ab833, by Abcam (1 mentions)
Ab52917, by Abcam (1 mentions)
Ab97959, by Abcam (1 mentions)
P stat3 d3a7, by Cell Signaling Technology (1 mentions)
Ab86299, by Abcam (1 mentions)
Metamorph software, by Molecular Devices (1 mentions)
Mca771g, by Bio-Rad (1 mentions)
22 protocols using mca497r
1
Immunofluorescent Staining of Eye Tissues
2
F4/80 Immunostaining of NB Xenografts
3
Comprehensive Histology and Staining Protocol
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Tissues were fixed in 10% formalin, subjected to paraffin-embedded sectioning and stained with hematoxylin or immunostained with anti-UCP1 (Abcam, ab10983, 1:100) and anti-perilipin A (Abcam, ab61682. 1:100) antibodies followed by incubation with the secondary antibody conjugated with Alexa 488 (Invitrogen, A32814, 1:500) and 594 (Invitrogen, A32754, 1:500). TUNEL (terminal deoxynucleotidyl transferase dUTP-mediated nick end labeling) (Takara, cat. no. MK500), cleaved caspase-3 (Cell signaling, #9661, 1:100), Iba-1 (Wako, 019-19741, 1:200), and F4/80 (Serotec, MCA497R, 1:200) immunostaining were performed per manufacturer’s instructions. To assess hepatic steatosis, multiple slides of frozen liver sections fixed with 10% buffered formalin for 30 min at room temperature were stained for 7 min with a filtered solution of 0.7% Oil Red O in propylene glycol, and counterstained with hematoxylin for 1 min, washed 3 times with distilled water, and then visualized. Liver sections were also stained with azan to identify fibrosis. To estimate adipocyte size, tissues (iWAT and eWAT) were fixed overnight with neutral-buffered 10% formalin at 4 °C, paraffin embedded, sectioned, and stained with hematoxylin and eosin. Adipocyte area was determined by quantifying the adipocyte area of a total of 20000~ cells from 7–9 animals per study group using Adiposoft image analysis software.
4
Immunohistochemical Analysis of Lung Tumor Markers
5
Macrophage Immunohistochemical Quantification
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Immunohistochemical detection of macrophages was performed as previously described
[10 (link)]. Immunostaining was performed by using a rat anti-mouse F480 monoclonal primary antibody (1:200, MCA497R; Serotec, Oxford, UK) and biotinylated goat anti-rat IgG (1:100, AP183β; Millipore, Billerica, MA, USA). For analysis, 20 to 25 sequential fields of view (200× magnification) were captured and Metamorph software (Molecular Devices, Sunnyvale, CA, USA) was used to determine total numbers of nuclei and F4/80+ cells.
[10 (link)]. Immunostaining was performed by using a rat anti-mouse F480 monoclonal primary antibody (1:200, MCA497R; Serotec, Oxford, UK) and biotinylated goat anti-rat IgG (1:100, AP183β; Millipore, Billerica, MA, USA). For analysis, 20 to 25 sequential fields of view (200× magnification) were captured and Metamorph software (Molecular Devices, Sunnyvale, CA, USA) was used to determine total numbers of nuclei and F4/80+ cells.
6
Analyzing Macrophage Infiltration in Aortic Plaques
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Mice were euthanized (n = 3–4/group), perfused with PBS (5 mL) and the aortic arch with heart tissue was embedded in OCT freezing compound. Seven-micrometer serial cryosections were collected every 70 µm in the innominate artery. Immunohistochemistry was performed on cryosections corresponding to greatest plaque accumulation in the innominate artery as previously described [42] (link), [53] (link) using rat anti-mouse F4/80 (no. MCA497R; Serotec, Oxford, U.K.) or isotype controls (no. MCA1125; Serotec). Biotinylated anti-rat (mouse absorbed) IgG was used as secondary Ab (Vector Laboratories, Burlin- game, CA). Nuclei were counter-stained with hematoxylin. Digital micrographs were captured at 10× and 40×.
7
Immunohistochemical Profiling of Tumor Microenvironment
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Tumor tissue was harvested in 4% paraformaldehyde overnight, paraffin-embedded and sectioned at 6 μm. Detection of macrophages and neutrophils was achieved after trypsin antigen retrieval using a primary antibody targeted against respectively F4/80 clone Cl:A3-1 (MCA497R, AbD Serotec, Kidlington, UK) and Ly6B2 clone7/4 (MCA771G, AbD Serotec, Kidlington, UK) at a dilution of 1:100 in phosphate-buffered saline (PBS)/0.1% Tween. Blood vessels were detected using an antibody against Endomucin clone V.7C7 (sc-65495, Santa Cruz Biotechnology, Dallas, TX, USA) at a dilution of 1:500 in PBS/0.1% Tween-20. Unspecific binding of primary antibody was prevented by blocking the sections in PBS/0.1% Tween-20 with 10% normal donkey serum. Visualization was obtained using a donkey-anti-rat Alexa594-labeled secondary antibody at 1:500 dilution in PBS/0.1% Tween-20. Sections were mounted using Prolong Gold Antifade with DAPI solution (Invitrogen, Carlsbad, CA, USA). The whole section on a slide was imaged at 10x magnification using automated slide scanning and the Zeiss Axiovision plugin MosaiX (Carl Zeiss, Jena, Germany) to stitch the images together, followed by image processing in Fiji and analysis on Volocity.
8
Histological and Immunohistochemical Analysis of Tumor Tissue
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Non-radioactive tumors were resected on day 7, fixed in 10% formalin for 48 h, and then transferred to 70% ethanol for immediate histological processing. Radioactive tumors were resected on day 7, fixed in 10% formalin, decayed for 3 months, and then transferred to 70% ethanol for histological processing. Tumor tissue was embedded in paraffin, and 4-μm sections were prepared. Tissue sections were processed for hematoxylin staining and IHC using Automated Leica Bond Rx stainer with Bond Refine Polymer Detection kit (Leica, DS9800). All antibodies were diluted in IHC/ISH Super Blocker (Leica, PV6199). Primary antibodies and working dilutions using HIER Retrieval Buffer 2 (Leica, AR9640) were as follows: CD3 (1:150; Abcam, ab16669), CD4 (1:800; eBio, 14-9766), and CD8a (1:1000; eBio, 14-0808). For F4/80 (1:500; AbD Serotec, MCA497R), an Enzyme 1 pre-treatment was performed before staining with antibody (AR9551). Antibodies CD4, CD8a, CD19, and F4/80 all required a secondary antibody before polymer detection using Rabbit anti-rat (Vector labs BA4001) at a dilution of 1:100. Immunohistochemistry slides were digitalized using the Olympus VS120-L100-W automated slide scanner (Shinjuku, Tokyo, Japan). They were batch-scanned on the brightfield setting at 20× magnification. The color camera used was the Pike 505C VC50.
9
Immunohistochemical Analysis of Macrophages
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For antigen retrieval, sections were dewaxed, rehydrated, and warmed with a citrate buffer. Next, to block endogenous peroxidase, they were incubated with peroxide. Investigators stained the sections with anti-F4/80 antibody (1/300 dilution; MCA497R, Serotec, Raleigh, NC, USA) overnight at 4°C after blocking with goat serum. The next day, the immunoperoxidase technique, using match application, with Envision kits (Boster Biological Technology, China) was performed. These samples were then stained with diaminobenzidine and hematoxylin. The positive stained cells of F4/80 of sections were counted using ×200 magnification.
10
Immunohistochemical Detection of F4/80 in Skin Lesions
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Sections (4 μm) of skin lesion tissue from mice were mounted on glass slides, stained with 3,3-diaminobenzidine tetrahydrochloride (Dojindo, Kumamoto, Japan), and counterstained with hematoxylin and processed for immunohistochemical detection of F4/80 by mouse monoclonal F4/80 antibody (Serotec, MCA 497 R, Raleigh, NC) according to standard immunoperoxidase procedure.
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