For immunofluorescence double staining, quenching of endogenous peroxidase and blocking of endogenous biotin were omitted. Sections were incubated with anti-TTF1 SP141 (1:200; Abcam ab227652) and anti-P40(1:1000; a gift from Dr. Philip J Coates, Masaryk Memorial Cancer Institute, Czech Republic); or with anti-MMP12 (1:100; Novus Bio, NBP2-67344) and anti-F4/80 (1:100; Bio-Rad, MCA497GA); or with anti-IL-1β (1:100; abcam, ab283818) and anti-F4/80( 1:100; Bio-Rad, MCA497GA); or with anti-Arg1(1:100; Santa Cruz, sc-18351) and anti-F4/80 (1:1000; Bio-Rad, MCA497GA); or with anti-IL-1β (1:100; abcam, ab283818) and anti-Arg1(1:100; Santa Cruz, sc-18351). Primary antibodies were detected with donkey anti-rabbit FITC (1:400; Invitrogen, A21206) and donkey anti-mouse 594 (1:400; Invitrogen, A21203) or donkey anti rabbit FITC (1:400; Invitrogen, A21206) and donkey anti-rat 594 (1:400; Invitrogen, A21209); donkey anti rabbit FITC (1:400; Invitrogen, A21206) and donkey anti-goat Cy3 (1:400; Jackson ImmunoResearch, 05-165-147). Samples were covered with medium containing with 4', 6'-diamidino-2-phenylindole (DAPI) (Vector) for cell nuclei visualization. The Keyence microscope (BZ-X810) was used to take the images.
Mca497ga
Manufactured by Abcam
MCA497GA is an immunoglobulin G (IgG) monoclonal antibody that recognizes the human CD47 antigen. CD47 is a cell surface protein involved in the regulation of phagocytic activity of macrophages. This antibody can be used in various applications such as flow cytometry, immunohistochemistry, and Western blotting.
Automatically generated - may contain errors
Lab products found in correlation
Ab25877, by Abcam (2 mentions)
Af114, by R&D Systems (2 mentions)
T7451, by R&D Systems (2 mentions)
Prb 160p, by Bio-Rad (2 mentions)
Eclipse nie, by Leica (2 mentions)
Ab8251, by Abcam (1 mentions)
Ab133557, by Abcam (1 mentions)
Rm2255 microtome, by Leica (1 mentions)
St5010, by Leica (1 mentions)
Pannoramic 250 slide scanner, by 3DHISTECH (1 mentions)
Ab227652, by Abcam (1 mentions)
A21203, by Thermo Fisher Scientific (1 mentions)
A21209, by Thermo Fisher Scientific (1 mentions)
Sc 18351, by Santa Cruz Biotechnology (1 mentions)
A21206, by Jackson ImmunoResearch (1 mentions)
A21206, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Bz x810, by Keyence (1 mentions)
Ab15580, by Abcam (1 mentions)
Ab81289, by Thermo Fisher Scientific (1 mentions)
6 protocols using mca497ga
1
Multiplex Immunofluorescence Staining Protocol
2
Histological Analysis of HGSC Tissue
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Tissue samples were fixed in 10% Formalin and embedded in paraffin wax (Leica RM2255 microtome). Sections were cut in Palm slides and underwent Haematoxylin&Eosin (H&E) staining and automated Immunohistochemistry staining (Leica ST5010). Histology slides were imaged on a Pannoramic 250 Slide Scanner (3DHISTECH) and images exported to ImageJ. Images were converted to RGB stacks and a threshold was set across all images to identify positive staining. H&E and PAX8 staining was used to identify HGSC tissue. Regions of interest (ROIs) were then manually drawn around discrete areas of HGSC tissue. The data were reported as the total area percentage of the ROI staining positive. PAX8: Abcam ab13611, 1:100; F4/80: Biorad MCA497GA, 1:1000; Adenovirus: Abcam ab8251, 1:1000; DX5: Abcam ab133557, 1:250.
3
Multiparametric Immunostaining Protocol
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Paraffin slides were rehydrated by consecutive, descending
processing through Xylene and Ethanol (100%–50%). Paraffin slides
underwent citrate-based retrieval (Vector Unmasking), for frozen sections
only select panels were citrate retrieved.
Tissue sections were blocked and permeabilized for 30 – 60
minutes in 10% NDS, 4% BSA and 0.5% TritonX. Primary antibodies (Krt5 (rb)
Covance (Prb-160P) 1:1000; F4/80 (rt) Biorad (MCA497GA) 1:250; RFP (gt)
Abcam (ab25877) 1:500; acet.Tub (ms) Sigma (T7451) 1:2000; CD45 (gt) R&D
(AF114) 1:250) were diluted in primary block solution (2.5% NDS, 1% BSA,
0.125% TritonX) and incubated overnight at 4°C in a humidified
chamber. All fluorophore-conjugated secondary antibodies were diluted in
secondary block solution (5% NDS, 2% BSA, 0.25% TX) at 1:500 containing
Höechst nuclear stain dye. EdU staining was performed according to
manufacturer recommendations (Invitrogen). Images of sections were captured
on a Nikon Eclipse NiE or a Leica SP6.
processing through Xylene and Ethanol (100%–50%). Paraffin slides
underwent citrate-based retrieval (Vector Unmasking), for frozen sections
only select panels were citrate retrieved.
Tissue sections were blocked and permeabilized for 30 – 60
minutes in 10% NDS, 4% BSA and 0.5% TritonX. Primary antibodies (Krt5 (rb)
Covance (Prb-160P) 1:1000; F4/80 (rt) Biorad (MCA497GA) 1:250; RFP (gt)
Abcam (ab25877) 1:500; acet.Tub (ms) Sigma (T7451) 1:2000; CD45 (gt) R&D
(AF114) 1:250) were diluted in primary block solution (2.5% NDS, 1% BSA,
0.125% TritonX) and incubated overnight at 4°C in a humidified
chamber. All fluorophore-conjugated secondary antibodies were diluted in
secondary block solution (5% NDS, 2% BSA, 0.25% TX) at 1:500 containing
Höechst nuclear stain dye. EdU staining was performed according to
manufacturer recommendations (Invitrogen). Images of sections were captured
on a Nikon Eclipse NiE or a Leica SP6.
4
Multiparametric Immunostaining Protocol
5
Immunohistochemistry and Immunofluorescence Protocols
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Primary antibodies used for immunohistochemistry were α‐keratin 14 (Covance, PRB‐155P, 1:50,000), and α‐CD31 (ScyTek, DIA‐310, 1:50). Primary antibodies used for immunofluorescence were α‐F4/80 (Bio‐Rad, MCA497GA, 1:500) and α‐Ki67 (Abcam, ab15580, 1:1000). Secondary antibodies used for immunohistochemistry were biotinylated α‐rat and biotinylated α‐rabbit (Vector Laboratories, Burlingame, CA, USA). Secondary antibodies used for immunofluorescence were alexafluor 555‐conjugated α‐rabbit IgG and α‐rat IgG (Invitrogen).
6
B16Bl6 Melanoma Lung Metastasis Assay
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B16Bl6 melanoma lung metastasis B16Bl6 melanoma cells were cultured in complete RPMI medium and intravenously transferred to mice (2x10 5 cells per mouse). After 12 days, the mice were sacrificed and lungs were placed in Fakete's fixation solution. Metastatic nodules were enumerated in the stereoscope and lung sections were later processed for H&E staining and immunohistochemistry using antibodies against CD3 (Abcam, ab16669), B220 (BD Biosciences, 553084), F4/80 (Serotec, MCA497GA), CD34 (Abcam, ab81289), claudin-5 (Invitrogen, 35-2500) and VE-cadherin (eBioscience, 14-1441). The in vivo siRNA experiments were performed with DACC lipoplexes designed by Silence Therapeutics (Fehring et al., 2014) . The siRNAs were manufactured by Biospring/Frankfurt and the sequences are reported at Table S2. The DACC siRNA formulations were administered intravenously (via tail vein) at a dose of 0.084mg/mouse at days 3, 5 and 7 post B16Bl6 intravenous delivery.
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