Anti-CD45 抗体

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD45 antibody (AB10558)

Immunohistochemical analysis of formalin fixed paraffin embedded human tonsil labelling CD45 with ab10558 at a concentration of 0.5µg/ml. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with a Bond™ Polymer Refine Detection kit. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution 2) for 20mins. ab10558 anti-CD45 antibody was incubated for 15mins at room temperature. Sections were counterstained with Hematoxylin. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD45 antibody (AB10558)

IHC image of CD45 antibody staining in a section of formalin-fixed paraffin-embedded normal human spleen* performed on a Leica BOND^TM system using the standard protocol. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6 epitope retrieval solution 1) for 20mins. The section was then incubated with ab10558 1ug/ml for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank supported by the NIHR Cambridge Biomedical Research Centre

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD45 antibody (AB10558)

Immunohistochemical analysis of formalin fixed paraffin embedded human tonsil labelling CD45 with ab10558 at a concentration of 0.1µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins. ab10558 anti-CD45 antibody was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• Flow Cyt (Intra)

AbReview12835****

Flow Cytometry (Intracellular) - Anti-CD45 antibody (AB10558)

Asynchronous KM-H2 cells were pelleted and labeled by indirect immunofluorescence. Cells were stained with ab10558 (1/200 dilution) for 30min at 4'C washed and then stained with goat anti-rabbit alexafluor 488 (1/200 dilution). Forward/Side scatter were used to eliminate cellular debris. The accompanying marker was applied such that only 2% of the IgG control was positive Based on the accompanying image approximately 8.4% of cells exhibited positive staining for anti-CD45. Since KM-H2 are known to have low levels of CD45 transcripts they are expected to have low levels of CD45 which is reflected in the ~8%. This image is from an Abreview.

This image is courtesy of an abreview submitted by Kirk Mcmanu.

• IHC-P

PubMed

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD45 antibody (AB10558)

ab10558 staining CD45 in Human tonsil tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Negative control is shown in panel. Blocking was with horse serum (1/75) for 1 hour at room temperature. Samples were incubated with primary antibody (1/10) overnight at 4°C. A Biotin-conjugated Horse anti-mouse polyclonal (1/200) was used as the secondary antibody.

Lorenzi T et al., PLos One 7:e35232 (2012), Fig 4, doi: 10.1371/journal.pone.0035232 Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-CD45 antibody (AB10558)

Overlay histogram showing Jurkat cells stained with ab10558 (red line). The cells were fixed with 80% methanol (5 min) and incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab10558 1μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/1000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5000 events was performed.

Please note that Abcam do not have any data for use of this antibody on non-fixed cells. We welcome any customer feedback.

• IHC-P

AbReview50074****

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD45 antibody (AB10558)

Immunohistochemical analysis of formaldehyde fixed human cephalic sections. Primary antibody ab10558 to CD45 incubated at a concentration of 1/100 for 4°C for 18 hours. Secondary antibody used was a goat anti-rabbit congugated to biotin at a 1/200 dilution. Blocking was done with serum at a 10% concentration for 1 hour at 25°C.

This image is courtesy of an anonymous abreview.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD45 antibody (AB10558)

ab10558 (1 : 40 dilution) staining CD45 in paraffin-embedded human tonsil (left panel) using an automated system (Ventana Discovery). Right-hand panel shows negative control (no primary antibody).
Using this protocol there is strong membrane staining of B cells in the germinal centres and mantle zone of the follicles and scattered cells of the interfollicular areas (paracortical T and B cells). There is a mild to moderate degree of cytoplasmic staining associated with the membrane staining in these specific cells.

Sections were rehydrated and antigen retrieved in CC1 Cell Conditioning Buffer using Ventana Extended Retrieval programme. Slides were blocked in 3% H₂O₂ /4 min/ 37°C and incubated with ab10558 (1 : 40 dilution / 1 hour/ 37°C). Sections then blocked (4mins/ 37°C) and incubated with Dako swine anti-rabbit antibody (1 : 50 dilution, 28 min/ 37°C). Staining was amplified and detected by incubation with Ventana Streptavidin ABC (HRP-DAB) system (16 min/ 37°C) before being counterstained with hematoxylin.

• WB

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Western blot - Anti-CD45 antibody (AB10558)

Secondary antibody - goat anti-rabbit H&L (HRP) (ab6721)

All lanes:

Western blot - Anti-CD45 antibody (ab10558) at 1/500 dilution

Lane 1:

Jurkat Whole Cell Lysate at 20 µg

Lane 2:

Jurkat Whole Cell Lysate at 20 µg with Human CD45 peptide (ab17553)

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab6721'>ab6721</a>) at 1/5000 dilution

Predicted band size: 147 kDa

false

Exposure time: 3min

• WB

Ap29663****

Western blot - Anti-CD45 antibody (AB10558)

This blot was produced using a 3-8% Tris Acetate gel under the TA buffer system. The gel was run at 150V for 60 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab10558 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.

CD45 contains a number of potential glycosylation sites (SwissProt) which may explain its migration at a higher molecular weight than predicted.

All lanes:

Western blot - Anti-CD45 antibody (ab10558) at 1 µg/mL

Lane 1:

Jurkat (Human) Whole Cell Lysate at 20 µg

Lane 2:

RAW 264.7 (Mouse leukaemic monocyte macrophage cell line) Whole Cell Lysate at 20 µg

Lane 3:

Spleen (Mouse) Tissue Lysate at 20 µg

Lane 4:

Spleen (Rat) Tissue Lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG H&L (HRP) at 1/50000 dilution

Predicted band size: 147 kDa

Observed band size: 190 kDa,230 kDa

true

Exposure time: 1min

• IHC

CiteAb

Immunohistochemistry - Anti-CD45 antibody (AB10558)

Immunohistochemistry using Anti-CD45 antibody, ab10558. Publication image from Gerzanich, V. et al., 2015, J Neuroinflammation, 26581714. Legend direct from paper.

Glibenclamide and Abcc8-/- suppress immune cell infiltration in EAE. a–d White matter of lumbar spinal cord sections from WT control (a), untreated pid-30 WT/EAE (b), glibenclamide-treated pid-30 WT/EAE (c), and pid-30 Abcc8-/-/EAE (d) mice, stained with H&E or immunolabeled for CD45 (leucocyte), CD3 (T cells), CD20 (B cells), or CD11b (macrophage/microglia), as indicated; original magnification, x200 (H&E) or x400 (all immunolabelings). eleft panel : percent of quadrants with inflammatory cells on H&E; four mice/group. efour right panels : Quantification of CD-45-, CD3-, CD20-, and CD11b-expressing cells in white matter; four mice/group; ##P<0.01 with respect to WT control; **P<0.01, and ***P<0.001 with respect to WT/EAE; scale bars, 100 μm.

• IHC

CiteAb

Immunohistochemistry - Anti-CD45 antibody (AB10558)

Immunohistochemistry using Anti-CD45 antibody, ab10558. Publication image from Lachota, M. et al., 2018, Angiogenesis, 29332242. Legend direct from paper.

Histology and immunofluorescence in rat corneal sections. a Hematoxylin and Eosin (H&E), b VEGF-A (green); c CCL2 (green); d TNF-α (green); e CXCL5 (green); f CD45 (green) and g HIF-1α (green) staining in rat cornea tissue. Nuclear counterstaining by DAPI (blue) in fluorescent images.

• IHC-P

CiteAb

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD45 antibody (AB10558)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) using Anti-CD45 antibody, ab10558. Publication image from Gerzanich, V. et al., 2017, J Neuroinflammation, 28865458. Legend direct from paper.

Chronic phase glibenclamide reduces the inflammatory burden in EAE. White matter of lumbar spinal cord sections from non-EAE control (CTR), untreated EAE mouse (EAE), and EAE mouse treated with glibenclamide starting on pid-24 (EAE + G), examined at pid-40, stained with H&E or immunolabeled for CD45 (leukocyte), CD20 (B cells), or CD3 (T cells), as indicated; original magnification, x200 (H&E) or x400 (all immunolabelings); bar graphs : percent of quadrants with inflammatory cells on H&E; quantification of CD45+, CD20+, and CD3+ cells in white matter; 5 mice/group; ##P < 0.01 with respect to non-EAE control (CTR); **P < 0.01 with respect to untreated EAE; scale bars 100 μm.

• WB

CiteAb

Western blot - Anti-CD45 antibody (AB10558)

Western Blotting using Anti-CD45 antibody, ab10558. Publication image from Ferrara, M. et al., 2020, Front Immunol, 32670275. Legend direct from paper.

Redox modulation of HMGB1 in spleen and in drug-intoxicated liver. (A) Western blot probed with anti-CD45, anti-HMGB1, and anti-GAPDH antibodies in reducing conditions (upper panels) or probed with anti-HMGB1 antibody in non-reducing conditions (lower panel) on lysates of spleen and liver isolated from control WT mice. In the lower panel, the upper band corresponds to the fully reduced-HMGB1 (frHMGB1) and the lower band to the disulphide-HMGB1 (dsHMGB1). (B,C) Quantification of total CD45 and HMGB1 protein levels normalized on GAPDH (B), and HMGB1 redox isoforms percentage (C) in spleen and liver lysates. A.U. = arbitrary unit (n = 4 mice/group). (D–G) Drug-induced liver injury (DILI) was induced by i.p. injection of acetaminophen (APAP), 300 mg/kg (body weight). Serum collection and necroscopy were performed at the indicated time points. (D) Representative images of DAPI and Evans Blue (EB) staining, Haematoxylin & Eosin (H&E) staining, and CD45 and HMGB1 immunostaining in liver sections from control mice (Ctrl) and at days 1, 2, 3, and 7 after DILI. Scale bars, 50 μm. (E) Alanine aminotransferase (sALT) and HMGB1 levels in serum before and after APAP injection in mice (n ≥ 5 mice/group). (F) Quantification of total number of intrahepatic leukocytes (IHLs) in control mice and at days 1 and 2 post-APAP injection (n = 4 mice/group). (G) Quantification of HMGB1 redox isoforms percentage, from Western blot assays performed in non-reducing conditions with anti-HMGB1 antibody, in IHLs isolated from control mice and at days 1 and 2 post-APAP injection (n = 4 mice/group). Data represent the means ± SEM and statistical significance was calculated by Student T-test (B), One-way (E,F) and Two-way ANOVA (C,G). ***p < 0.001; ****p < 0.0001; ns, not significant.

false

• WB

CiteAb

Western blot - Anti-CD45 antibody (AB10558)

Western Blotting using Anti-CD45 antibody, ab10558. Publication image from Lachota, M. et al., 2018, Angiogenesis, 29332242. Legend direct from paper.

Quantitative qRT-PCR and Western blot analysis of inflammatory and neovascular factors in suture-stimulated rat corneas at 96 h. Quantitative qRT-PCR analysis of aVegf-a, bCxcl5, cCcl2, and dCxcr2 expression in rat cornea. (n = 5) One-way ANOVA test with Tukey multiple comparisons were used to determine statistical significance. e Western blot analysis of CD45, phospho-IκBα and VEGF-A expression in rat cornea lysate. β-Actin used as a loading control. n.s. p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001.

false