Tissues were fixed in 10% formalin for paraffin embedding. Tibias were decalcified in 14% EDTA prior to paraffin embedding. 5µm sections were stained by immunohistochemistry (IHC) with rat anti-mouse CD45 (BD Pharmingen #550539), CD3 (Dako #A0452), B220 (BD Pharmingen #550286). Flag-tagged HBZ expression was detected following citrate antigen retrieval with the anti-flag antibody (Sigma #F1804, 5ug/ml) and detected using the HistoMouse-Plus Broad Spectrum (AEC) kit (Life Technologies, catalog #849541). Tibias were stained with the tartrate-resistant acid phosphatase (TRAP) (Sigma-Aldrich) to visualize osteoclasts. The number and surface area of TRAP+ cells/ bone surface area were quantified at 5X magnification for WT (n = 7) and HBZ (n = 7) mice. Images were acquired using the NanoZoomer 2.0-HT System (Hamamatsu Photonics) or the Zeiss Axio Scan Z1.
Rat anti mouse cd45
Manufactured by BD
Sourced in United States
Rat anti-mouse CD45 is a primary antibody that recognizes the CD45 cell surface antigen expressed on mouse leukocytes. CD45 is a receptor-linked protein tyrosine phosphatase that plays a critical role in the regulation of T-cell and B-cell antigen receptor signaling.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (3 mentions)
Rabbit anti mouse lyve 1, by Abcam (2 mentions)
Donkey anti rabbit 488, by Jackson ImmunoResearch (2 mentions)
Donkey anti rabbit tritc, by Jackson ImmunoResearch (2 mentions)
Armenian hamster anti mouse cd31, by Merck Group (2 mentions)
Goat anti hamster tritc, by Jackson ImmunoResearch (2 mentions)
Goat anti rat alexa flour 647, by Jackson ImmunoResearch (2 mentions)
Rabbit anti mouse fgf 2, by Abcam (2 mentions)
Fitc conjugated avidin, by BioLegend (2 mentions)
Rat anti mouse cd11b, by BD (2 mentions)
Axio scan z1, by Zeiss (1 mentions)
Anti flag antibody, by Merck Group (1 mentions)
Nanozoomer 2.0 ht system, by Hamamatsu Photonics (1 mentions)
Mouse anti human cd34, by BD (1 mentions)
Mouse anti human cd45, by BD (1 mentions)
7 aminoactinomycin d, by Merck Group (1 mentions)
Annexin 5 cy5, by BD (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Sorafenib, by Selleck Chemicals (1 mentions)
Rat anti mouse sca1, by BD (1 mentions)
17 protocols using rat anti mouse cd45
1
Immunohistochemical Characterization of Bone Inflammation
2
Immunofluorescent Analysis of Corneal Vasculature
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Corneas were excised after cardiac perfusion with cold PBS of deeply anesthetized mice. The corneas were fixed in 4% paraformaldehyde (PFA) for 30 min at 4° C followed by three 15 min washes with 1% PBS in 1% Triton X-100 (Sigma) at room temperature (RT). The corneas were blocked with 10% normal donkey serum overnight at 4° C. For primary staining, the corneas were incubated overnight with a cocktail of rabbit anti-mouse LYVE-1 (Abcam), armenian hamster anti-mouse CD31 (Millipore), and rat anti-mouse CD45 (BD Pharmingen) in 0.1% PBS in 0.1% Trition X-100. For secondary staining, the corneas were incubated overnight with a cocktail of donkey anti-rabbit 488, goat anti-hamster TRITC, and goat anti-rat alexa flour 647 (all from Jackson Immunoresearch) in 0.1% PBS in 0.1% Trition X-100. The corneas were washed 5X, 30 min per wash in 0.1% PBS in 0.1% Trition X-100 at RT, and mounted on a glass slide after making radial cuts. For mast cell and FGF-2 co-staining, rabbit anti-mouse FGF-2 (Abcam) was used as primary antibody followed by an overnight incubation in a cocktail of donkey anti-rabbit TRITC (Jackson Immunoresearch) and FITC-conjugated Avidin (Biolegend). The corneas were then imaged with a laser-scanning confocal microscope, IX-81, FV500 (Olympus).
3
Evaluation of Hypoxia-Targeting Agents
4
Immunofluorescent Analysis of Corneal Vasculature
5
Characterization of Mouse Mesenchymal Stem Cells
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The cell surface markers of MSCs were analyzed using a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA). Briefly, cells that reached 90% confluence were harvested using 0.25% EDTA and washed twice in Dulbecco’s phosphate buffered saline supplemented with 10% FBS. The cells for detecting CD11b, CD34, CD45, Sca-1, CD44 and CD73 were labeled directly with BB515 or PE-conjugated CD markers (rat anti-mouse CD11b [1: 100, BD Pharmingen; BD Biosciences, Franklin Lake, NJ, USA], rat anti-mouse CD34 [1: 100, BD Pharmingen], rat anti-mouse CD45 [1: 100, BD Pharmingen], rat anti-mouse Sca-1 [1: 100, BD Pharmingen], rat anti-mouse CD44 [1: 100, BD Pharmingen], rat anti-mouse CD73 [1: 100, BD Pharmingen]).
6
Immunohistochemical Analysis of Mouse Retina
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Eyes were harvested from mice and fixed in 4% paraformaldehyde for 5 hours at room temperature. For cryopreservation, fixed eye samples were placed in sucrose gradients 10%, 15%, and 20% for 1 hour each at room temperature. Eye samples were placed in an OCT compound and frozen at −80°C. The 10-μm thick eye sections were cut using a cryostat. Sections were blocked with 10% normal donkey serum with 0.5% Tween-20 for 1 hour. Sections were stained with primary antibodies (rabbit polyclonal anti-iNOS [Santa Cruz Biotechnology, CA, USA], rabbit polyclonal anti-COX2 [Abcam, Cambridge, MA, USA], and rabbit polyclonal immunoglobulin G (IgG) control [Jackson Immunoresearch, West Grove, PA, USA]) at 1:650 dilution at 4°C overnight. To detect CD45+ cells, rat antimouse CD45 (1:500 dilution; BD Biosciences) was added to the sections at 4°C overnight. Sections were stained with secondary antibodies, fluorescein isothiocyanate goat antirabbit (Jackson Immunoresearch) at 1:100 dilution and DI594 goat antirat IgG (Jackson Immunoresearch) at 1:10000 dilution for 90 minutes. Hoechst deoxyribonucleic acid dye (Thermo, Rockford, IL, USA) at 1:1000 was used to stain nuclei. Retinas were imaged using a fluorescence microscope.
7
Muscle Fibrosis and Regeneration Assessment
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On days 7, 14, and 28 after injury, mice were sacrificed and damaged muscle samples were harvested. Hematoxylin–Eosin (H&E) and Masson’s Trichrome staining were performed to detect fibrosis of injured limbs. Immunostaining was performed to determine the therapeutic effects of the PGE2 matrix. For immunostaining, primary antibodies were used as follow, mouse anti-mouse α-SMA (1:200, BD, USA), mouse anti-mouse Ki-67 (1:200, Invitrogen, USA), rabbit anti-mouse cleaved caspase-3 (1:100, Wanleibio, China) and rat anti-mouse F4/80 (1:100, Abcam, USA), anti-MyoD1 (1:1000, Wanleibio, China), rat anti-mouse CD45 (1:200, BD, USA). Secondary antibodies, Alexa Fluor 594 labeled goat anti-rat, Alexa Fluor 555 labeled goat anti-mouse, Alexa Fluor 488 labeled goat anti-mouse and Alexa Fluor 488 labeled goat anti-rabbit IgG were used. Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Immunohistochemistry was developed using a DAB peroxidase substrate DAB staining kit (ZSGB-BIO, China) according to the manufacturer’s instructions. The images were analyzed using ImageJ software.
8
Immunopanning Purification of Brain Endothelial Cells
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Brain endothelial cells from young and aged mice were purified by immunopanning97 (link) for further quantitative PCR and western blot assays. Mouse brains (n = 3 from each group for each experiment) were dissected, pooled, and diced into ~1 mm3 pieces. Tissues were dissociated into single cells by Neural Tissue Dissociation Kit (P; 130-092-628, Miltenyi Biotec, US). The cells were filtered with 70-µm cell strainer (#352350, Falcon, US) and were further treated with myelin removal beads (130-096, Miltenyi, Germany) to deplete myelin debris and myelin-associated cells. For immunopanning, rat anti-mouse CD45 (550539, BD Pharmingen, US, 1:1500 dilution), and rat anti-mouse CD31 (553370, BD Pharmingen, US, 1:250 dilution) were employed for negative and positive selection respectively. Cells adhered to positive selection dishes were dissociated by trypsinization and harvested.
9
Cardiac Graft Rejection and CAV Analysis
10
Immunofluorescence Staining of Mouse Cells
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Rat anti-mouse CD54 (intracellular adhesion molecule 1 (ICAM-1)) antibody (clone #: YN1/1.7.4, rat IgG2b, κ) was purchased from BioLegend (San Diego, CA, U.S.A.). Rat anti-mouse CD45 (clone #: 30-F11) was obtained from BD Biosciences Pharmingen (San Diego, CA, U.S.A.). Rabbit anti-GFP (ab6556) was purchased from Abcam (Cambridge, MA, U.K.). Alexa Fluor® 594 Donkey anti-Rat IgG, Alexa Fluor®488 Donkey anti-Rabbit IgG and Alexa Fluor®488 phalloidin were sourced from Molecular Probes (Invitrogen, U.S.A.). Recombinant mouse IL-1β and MCP-1 were purchased from PROSPEC (East Brunswick, NJ, U.S.A.). Recombinant human fibroblast growth factor 2 (rhFGF-2) was obtained from Miltenyi Biotec (Bergisch-Gladbach, Germany).
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