Anti-CD45 antibody [MEM-28]

9 product images

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  Flow Cytometry - Anti-CD45 antibody [MEM-28] (ab8216)

  Flow cytometry analysis (surface staining) of human peripheral blood cells with ab8216 (1 ug/ml), GAM-APC.

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  Image from Nel, Ivonne et al. PLoS ONE 11.4 (2016): e0153018. doi: 10.1371/journal.pone.0153018 Fig 3.

  Immunocytochemistry/ Immunofluorescence - Anti-CD45 antibody [MEM-28] (ab8216)

  Immunocytochemistry/ Immunofluorescence analysis of CTC isolated from mRCC patients stained for hematopoietic cells labeling CD45 with ab8216 (green). The cells were fixed in 4.5% paraformaldehyde for 15 min, washed in PBS, permeabilized with 1x Perm/Wash for 10 min, washing in PBS, blocking of unspecific antibody reactions by incubation with blocking solution containing 5% BSA for 30 min, binding of Anti-CD45 antibody [MEM-28] (ab8216) (final concentration: 5 μg/ml) overnight at 4°C, wash in 0,1% Tween, binding of secondary antibody (Cy3-conjugated goat anti-mouse) for 30 min at 37°C, washing in 0,1% Tween. Subsequently, cells were stained with DAPI for 10 min, mounted with anti-fading medium and stored in the dark until evaluation. Left: CD45, right: DAPI, bottom: merge.

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  Image from Nel, Ivonne et al. PLoS ONE 11.4 (2016): e0153018. doi: 10.1371/journal.pone.0153018 Fig 2. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

  Immunocytochemistry/ Immunofluorescence - Anti-CD45 antibody [MEM-28] (ab8216)

  Immunocytochemistry/ Immunofluorescence analysis of hematopoietic cells labeling CD45 with ab8216. The cells were fixed in 4.5% paraformaldehyde for 15 min, washed in PBS, permeabilized with 1x Perm/Wash for 10 min, washing in PBS, blocking of unspecific antibody reactions by incubation with blocking solution containing 5% BSA for 30 min, binding of Anti-CD45 antibody [MEM-28] (ab8216) (final concentration: 5 μg/ml) overnight at 4°C, wash in 0,1% Tween, binding of secondary antibody (Cy3-conjugated goat anti-mouse) for 30 min at 37°C, washing in 0,1% Tween.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD45 antibody [MEM-28] (ab8216)

  ab8216 staining human normal tonsil tissue. Staining is localised to cellular membranes.
  Left panel: with primary antibody at 1 ug/ml. Right panel: isotype control.
  Sections were stained using an automated system (DAKO Autostainer Plus ), at room temperature: sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffers citrate pH6.1 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.

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  Flow Cytometry - Anti-CD45 antibody [MEM-28] (ab8216)

  Overlay histogram showing peripheral blood lymphocytes stained with ab8216 (red line). The cells were incubated with the antibody (ab8216, 1μg/1x10⁶ cells) for 30 min at 4°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H&L) (Goat Anti-Mouse IgG H&L (DyLight® 488) preadsorbed ab96879) at 1/200 dilution for 30 min at 4°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (Mouse IgG1, Kappa Monoclonal [B11/6] - Isotype Control ab91353, 2μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed gating on peripheral blood lymphocytes.
  
  

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  Image from Weller, Patrick et al. PLoS ONE 9.12 (2014): e113706. doi: 10.1371/journal.pone.0113706. Fig 2. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

  Immunocytochemistry/ Immunofluorescence - Anti-CD45 antibody [MEM-28] (ab8216)

  Immunocytochemistry/ Immunofluorescence analysis of citrated peripheral blood hematologic cells taken from head and neck squamous cell carcinoma (HNSCC) patients labeling CD45 with ab8216 (green). The staining method included fixation of the cells in 4.5% paraformaldehyde for 15 min, washing in PBS, permeabilization with 1× Perm/Wash Buffer for 10 min, washing in PBS, blocking of unspecific antibody reactions by incubation with blocking solution containing 5% BSA for 30 min, binding of Anti-CD45 antibody [MEM-28] (ab8216) (final concentration: 5 μg/ml) overnight at 4°C, wash in 0,1% Tween, binding of Cy3-conjugated secondary antibody for 30 min at 37°C, washing in 0,1% Tween.

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  Immunocytochemistry/ Immunofluorescence - Anti-CD45 antibody [MEM-28] (ab8216)

  Immunocytochemistry/Immunofluorescence analysis of human peripheral blood mononuclear cells labelling CD45 (green) with ab8216 at 10 μg/mL. Nuclei were counterstained with DAPI (blue).

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  Flow Cytometry - Anti-CD45 antibody [MEM-28] (ab8216)

  Separation of human lymphocytes (red-filled) from CD45 negative blood debris (black-dashed) in flow cytometry analysis (surface staining) of human peripheral whole blood stained using ab8216 at 2 µg/ml (GAM APC).

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  Western blot - Anti-CD45 antibody [MEM-28] (ab8216)

  Western blotting analysis was performed on whole cell extracts (RIPA lysis buffer) of Jurkat, Raji, HeLa, and HEK293T cell lines, mixed and heated (100°C, 5 min) with reducing and non-reducing SDS-loading buffer. Samples were resolved using 7% Tris-glycine SDS gel electrophoresis.
  
  Nitrocellulose membrane blot was probed with ab8216 (1 µg/ml), followed by IRDye 800CW Goat-anti-Mouse IgG (green). Multiplex fluorescent Western blot detection was performed.
  
  CD45 molecules were detected at ~180-250 kDa in Jurkat and Raji cell lines.

  All lanes: Western blot - Anti-CD45 antibody [MEM-28] (ab8216)

  Lanes 1 and 5: Jurkat whole cell extracts with reducing SDS loading buffer

  Lane 2: Raji whole cell extracts with reducing SDS loading buffer

  Lane 3: HeLa whole cell extracts with reducing SDS loading buffer

  Lanes 4 and 8: HEK293T whole cell extracts with non-reducing SDS loading buffer

  Lane 6: Raji whole cell extracts with non-reducing SDS loading buffer

  Lane 7: HeLa whole cell extracts with non-reducing SDS loading buffer