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Rat anti mouse cd45 antibody

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Rat anti-mouse CD45 antibody is a laboratory reagent used for the identification and detection of CD45, a transmembrane protein tyrosine phosphatase expressed on the surface of most hematopoietic cells in mice.

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Lab products found in correlation

4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions) Biomag goat anti rat ig coupled magnetic beads, by Qiagen (2 mentions) Mouse anti epcam antibodies, by BioLegend (2 mentions) Dnase 1, by Merck Group (2 mentions) Pronase, by Roche (2 mentions) Anti rat 488 secondary ab, by Thermo Fisher Scientific (2 mentions) Rabbit anti cleaved caspase 3 antibody, by Cell Signaling Technology (1 mentions) Jnk inhibitor sp 600125, by Cayman Chemical (1 mentions) Tgf α, by R&D Systems (1 mentions) Rabbit anti phospho jnk antibody, by Cell Signaling Technology (1 mentions) Rat anti mouse f4 80 antibody, by Thermo Fisher Scientific (1 mentions) Rabbit anti jnk antibody, by Cell Signaling Technology (1 mentions) Mouse anti stat3 antibody, by Cell Signaling Technology (1 mentions) Rabbit anti phospho stat3 antibody, by Cell Signaling Technology (1 mentions) Rabbit anti ki67 antibody, by Abcam (1 mentions) Collagen coated, by MP Biomedicals (1 mentions) Mouse anti vimentin, by Santa Cruz Biotechnology (1 mentions) Dmem f12 media, by Thermo Fisher Scientific (1 mentions) Insulin, by Merck Group (1 mentions) Penicillin, by Merck Group (1 mentions)

7 protocols using rat anti mouse cd45 antibody

1

Inhibition of JNK Signaling in Immune Cells

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We purchased JNK inhibitor SP600125 from Cayman Chemical (Ann Arbor, MI, USA), and transforming growth factor alpha (TGF‐α) from R&D Systems (Minneapolis, MN, USA). Primary antibodies used in immunohistochemical and immunofluorescence assays included rabbit anti‐phospho‐JNK antibody (1:50; Cell Signaling Technology, Danvers, MA, USA), rat anti‐mouse CD45 antibody (1:50; BD Biosciences, San Jose, CA, USA), rat anti‐mouse F4/80 antibody (1:100; eBioscience, San Diego, CA, USA), rabbit anti‐Ki‐67 antibody (1:100; Abcam, Cambridge, UK), mouse anti‐α‐smooth muscle actin (α‐SMA) antibody (1:50; Santa Cruz, Santa Cruz, CA, USA), biotin hamster anti‐mouse CD11c antibody (1:100; BD Biosciences), rabbit anti‐cleaved caspase3 antibody (1:1600; Cell Signaling Technology) and rabbit anti‐CD8 antibody (clone EP1150Y, 1:250; Epitomics, Burlingame, CA, USA). Primary antibodies used in immunoblotting analysis included rabbit anti‐phospho‐JNK antibody (1:1000; Cell Signaling Technology), rabbit anti‐JNK antibody (1:1000; Cell Signaling Technology), rabbit anti‐phospho‐Stat3 antibody (1:2000; Cell Signaling Technology) and mouse anti‐Stat3 antibody (1:1000; Cell Signaling Technology).
Sato T., Shibata W., Hikiba Y., Kaneta Y., Suzuki N., Ihara S., Ishii Y., Sue S., Kameta E., Sugimori M., Yamada H., Kaneko H., Sasaki T., Ishii T., Tamura T., Kondo M, & Maeda S. (2017). c‐Jun N‐terminal kinase in pancreatic tumor stroma augments tumor development in mice. Cancer Science, 108(11), 2156-2165.
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2

Murine Airway Epithelial Cell Culture

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Mouse AEC cultures were prepared from female C57BL/6 mice as previously described22 (link), with minor modifications. Briefly, lungs from mice were digested in 1.5 mg/ml Pronase (Roche, USA), 0.1 mg/ml DNase I (Sigma-Aldrich, USA) for 1 hr at 37°C in 5% CO2. Single cell suspensions were incubated with purified rat anti-mouse CD45 antibody (BD Biosciences, USA) and epithelial cells negatively enriched with BioMag goat anti-rat Ig-coupled magnetic beads (Qiagen, USA). Cells were cultured on collagen-coated (MP Biomedicals, USA) plates in DMEM/F-12 media (Gibco, USA) supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and 5 μg/ml insulin (Sigma-Aldrich). To confirm cell purity, monolayers were detached with 3 mM EDTA and cells stained with mouse anti-EpCAM antibodies (BioLegend, USA) and examined by flow cytometry. Cell monolayers were negative for the presence of fibroblasts (anti-mouse vimentin (Santa Cruz, USA)). Monolayers were infected with the HKx31 strain of IAV (H3N2, MOI 1) in serum free DMEM/F-12 for 1 hr and then washed and incubated in DMEM/F-12 supplemented with 2% FCS and 4 μg/ml trypsin for 24 or 48 hrs.
Thomas B.J., Porritt R.A., Hertzog P.J., Bardin P.G, & Tate M.D. (2014). Glucocorticosteroids enhance replication of respiratory viruses: effect of adjuvant interferon. Scientific Reports, 4, 7176.
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3

Histological Analysis of Cardiac Remodeling

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After sacrifice, organs were rinsed in PBS and Zinc fixed for 48 hours (BD Pharmingen, #552658). 4 μm paraffin sections were cut to analyze histological changes of the left ventricle and septum. Collagen was stained with Picrosirius Red F3B (Klinipath, #80115) and interstitial collagen area was quantified after exclusion of vessels and endo- and epicardial connective tissue. Laminin staining was performed using rabbit anti-mouse laminin (Sigma, L9393, 1:100) and Vectastain Elite ABC kit (Vector laboratories) to assess cardiomyocyte size. Epicardial cardiomyocytes as well as longitudinal cells and cells without visible nucleus were excluded from cell size analysis. CD45 positive cells were stained with rat anti-mouse CD45 antibody (BD Pharmingen, #553076, 1:500) and Vectastain ABC-AP kit (Vector laboratories) and counted in the whole LV and septum. Capillaries were stained with biotinylated Griffonia (Bandeiraea) Simplicifolia Lectin I (Vector Laboratories, B-1105, 20 μg/mL) and Vectastain ABC-AP kit and cross-sectioned capillaries near the endocardium were counted. All analysis was performed in a blinded manner using a Leica DM2000 equipped with a Leica DFC450C camera and ImageJ software.
Peters T., Hermans-Beijnsberger S., Beqqali A., Bitsch N., Nakagawa S., Prasanth K.V., de Windt L.J., van Oort R.J., Heymans S, & Schroen B. (2016). Long Non-Coding RNA Malat-1 Is Dispensable during Pressure Overload-Induced Cardiac Remodeling and Failure in Mice. PLoS ONE, 11(2), e0150236.
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4

Immunohistochemical Labeling of Microglia

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Mice were sacrificed by carbon dioxide overdose and perfused with 4% paraformaldehyde (PFA). Brains and spinal cords were post-fixed in 4% PFA for 24h, and then cryoprotected in 30% sucrose solution for 2 days before being frozen in optimal cutting temperature compound. Cross, longitudinal and coronal sections of 10 μm thickness were blocked with phosphate buffered saline (PBS) containing 0.1% Triton X-100 for 1 hour. Sections were then incubated overnight at 4°C with rat anti-mouse CD45 antibody (BD, 1:1000). Bound antibody was detected with anti-rat 488 secondary Ab (Thermo Fisher Scientific) at a 1:200 dilution while DAPI (Thermo Fisher Scientific, 1:2000) incubation for 10 minutes at room temperature allowed for nuclear identification.
Hoghooghi V., Palmer A.L., Frederick A., Jiang Y., Merkens J.E., Balakrishnan A., Finlay T.M., Grubb A., Levy E., Gordon P., Jirik F.R., Nguyen M.D., Schuurmans C., Visser F., Dunn S.E, & Ousman S.S. (2020). Cystatin C Plays a Sex-Dependent Detrimental Role in Experimental Autoimmune Encephalomyelitis. Cell reports, 33(1), 108236.
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5

Histological and Immunohistochemical Staining Protocol

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Five-micron sections were deparaffinized and rehydrated in the following order for histological and immunohistochemical staining: three washes of xylene, 100% ethanol, 90% ethanol, 70% ethanol, and two washes of Tris-buffered vehicle. Slides were stained with hematoxylin and eosin for evaluation of morphology and picrosirius red to detect fibrillar collagen. Immunohistochemistry was performed using rat anti-mouse CD45 antibody (BD Biosciences), rabbit polyclonal CD68 antibody (Abcam), rabbit anti-mouse vimentin antibody (Cell Signaling), rabbit anti-mouse α-smooth muscle actin antibody (Abcam), anti-pan keratin antibody (Abcam), rabbit anti-collagen I antibody (Abcam), rabbit anti-collagen III antibody (Abcam), rabbit anti-FBN antibody (Abcam), and goat anti-mouse DCN antibody (R&D Systems, Minneapolis, MN, USA) as described previously10 (link). Histological evaluation was performed independently by two experimental pathologists who were blinded to the experimental conditions.
Shen Y., Zeglinski M.R., Turner C.T., Raithatha S.A., Wu Z., Russo V., Oram C., Hiroyasu S., Nabai L., Zhao H., Bozin T., Westendorf K., Kopko I., Huang R., Arns S., Tan J., Zeng H., Boey A., Liggins R., Jaquith J., Cameron D.R., Papp A, & Granville D.J. (2018). Topical small molecule granzyme B inhibitor improves remodeling in a murine model of impaired burn wound healing. Experimental & Molecular Medicine, 50(5), 68.
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6

Immunohistochemical Labeling of Microglia

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Mice were sacrificed by carbon dioxide overdose and perfused with 4% paraformaldehyde (PFA). Brains and spinal cords were post-fixed in 4% PFA for 24h, and then cryoprotected in 30% sucrose solution for 2 days before being frozen in optimal cutting temperature compound. Cross, longitudinal and coronal sections of 10 μm thickness were blocked with phosphate buffered saline (PBS) containing 0.1% Triton X-100 for 1 hour. Sections were then incubated overnight at 4°C with rat anti-mouse CD45 antibody (BD, 1:1000). Bound antibody was detected with anti-rat 488 secondary Ab (Thermo Fisher Scientific) at a 1:200 dilution while DAPI (Thermo Fisher Scientific, 1:2000) incubation for 10 minutes at room temperature allowed for nuclear identification.
Hoghooghi V., Palmer A.L., Frederick A., Jiang Y., Merkens J.E., Balakrishnan A., Finlay T.M., Grubb A., Levy E., Gordon P., Jirik F.R., Nguyen M.D., Schuurmans C., Visser F., Dunn S.E, & Ousman S.S. (2020). Cystatin C Plays a Sex-Dependent Detrimental Role in Experimental Autoimmune Encephalomyelitis. Cell reports, 33(1), 108236.
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7

Mouse Alveolar Epithelial Cell Isolation

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Mouse AEC cultures were prepared as described (Thomas et al., 2014 (link)), with minor modifications. Briefly, lungs were digested in 1.5 mg/mL Pronase (Roche) and 0.1 mg/ml DNase I (Sigma-Aldrich) for 60 min at 37°C in 5% CO2. Single cell suspensions were incubated with purified rat anti-mouse CD45 antibody (BD Biosciences) and epithelial cells negatively enriched using BioMag goat anti-rat Ig-coupled magnetic beads (Qiagen). Flow cytometry was used to confirm cell purity, which was approximately 95% with mouse anti-EpCAM antibodies (BioLegend) and cultures were on average 20% positive for podoplanin (AEC type I) and 70% for CD74 (AEC type II).
Kedzierski L., Tate M.D., Hsu A.C., Kolesnik T.B., Linossi E.M., Dagley L., Dong Z., Freeman S., Infusini G., Starkey M.R., Bird N.L., Chatfield S.M., Babon J.J., Huntington N., Belz G., Webb A., Wark P.A., Nicola N.A., Xu J., Kedzierska K., Hansbro P.M, & Nicholson S.E. (2017). Suppressor of cytokine signaling (SOCS)5 ameliorates influenza infection via inhibition of EGFR signaling. eLife, 6, e20444.
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