Anesthetized mice were euthanized, brains were sterilely dissected, and placed in ice-cold sterile PBS. Meninges within the skullcap were fixed in 4% paraformaldehyde (PFA) overnight and separated from the skullcap. Then the meninges were incubated in the block-perm buffer containing 2% normal goat serum, 1% BSA, 0.1% Triton X, and 0.05% Tween for 1 hr at room temperature with gentle rocking. The primary antibody anti-mouse CD8 (Novus, Cat# ABX-160A, 1:100), anti-lyve-1 (R&D Systems, Cat# AF2125), and the secondary antibody Alexa Fluor 488, 555 rabbit anti-mouse IgG, Alexa Fluor 555 rabbit anti-goat IgG (Life Technologies) were employed for CD8+ T cell and lymphatic vessel staining. The meninges were mounted with Prolong Gold with DAPI (Molecular Probes).
Anti mouse cd8
Manufactured by Novus Biologicals
Anti-mouse CD8 is a laboratory reagent used to detect and quantify CD8+ T cells in mouse samples. It binds specifically to the CD8 surface marker expressed on cytotoxic T cells.
Automatically generated - may contain errors
Lab products found in correlation
Prolong gold with dapi, by Thermo Fisher Scientific (1 mentions)
Anti lyve 1, by R&D Systems (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Anti mouse f4 80, by Bio-Rad (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Anti arginase 1, by GeneTex (1 mentions)
3 protocols using anti mouse cd8
1
Meningeal CD8+ T Cell Imaging
2
Immunohistochemical Analysis of ID8-luc Xenografts
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Tissue fixation, processing and sectioning methods of harvested ID8-luc xenografts were performed as previously described [18 (link),23 (link)]. In ID8-luc tumors, immunohistochemistry (IHC) for mib-1/Ki67 (1:1000; rabbit polyclonal anti-mib-1/Ki67, Vector Laboratories, Burlingame, CA), cleaved caspase-3 (1:300; rabbit polyclonal anti- cleaved caspase-3, Cell Signaling Technology, Danvers, MA), and γH2AX (Ser139) (1:100; mouse monoclonal MilliporeSigma) were performed as previously described [16] (link). Fluorescent-IHC image acquisition and analysis of co-staining for the pan-macrophage marker F4/80 (1:200 rat polyclonal anti-mouse F4/80, AbD Serotec, Oxford, UK), and the M2 macrophage marker arginase-1 (1:200; rabbit polyclonal anti-arginase-1, GeneTex, Irvine, CA), and separately for the cytotoxic T cell marker CD8 (1:100; rat monoclonal anti-mouse CD8, Novus Biologicals, Centennial, CO), was performed as previously described [23] (link). Cell nuclei were identified by staining with DAPI (Sigma-Aldrich). At least 1000 cells were counted in at least 5 independent fields under high power (x40) for these counts.
3
Immunohistochemical Analysis of Jejunum and Brown Adipose Tissue
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The jejunum and brown adipose tissues were fixed with 4% formalin and embedded in paraffin. Paraffinem-bedded sections (5 μm) deparaffinized, rehydrated, and steaming water 5 min, antigen retrieval with sodium citrate buffer (pH 6.0) for 6 min with pressure cooker. Endogenous catalase was inactivated with 3% hydrogen peroxide. Then, sections were incubated with anti-mouse CD4 (1:200, Novus, cat. NBP1-19371), anti-mouse CD8 (1:100, Novus, cat. NB200-578), anti-mouse UCP1 (1:100, ABclonal, cat. no A5857), and anti-mouse PGC-1α (1: 200, BOSTER, cat. no BA2816-1), overnight at 4 °C. Following morning, sections were incubated with the secondary antibodies in the incubator 40 min. After washed with distilled water and PBS, then sections were exposed to DAB, 1 min; and hematoxylin, 1 min; differentiate solution, 1 s; and observed after the neutral gum seal50 (link).
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