IHC image of HLA-DR [TAL 1B5] staining in Human skin melanoma formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab20181, 0.334µg/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.

All lanes : Anti-HLA-DR antibody [TAL 1B5] (ab20181) at 0.667 µg/ml

Lane 1 : Raji (Human Burkitt's lymphoma cell line) Whole Cell Lysate
Lane 2 : Daudi whole cell lysate (ab3951)
Lane 3 : Ramos (Human Burkitt's lymphoma cell line) Whole Cell Lysate
Lane 4 : Human spleen tissue lysate - total protein (ab29699)
Lane 5 : Tonsil (Human) Tissue Lysate
Lane 6 : Human liver tissue lysate - total protein (ab29889)

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 29 kDa
Observed band size: 29,35 kDa

why is the actual band size different from the predicted?


Exposure time: 16 minutes

Human peripheral blood lymphocytes stained with ab20181 (red line). Human whole blood was processed using a modified protocol based on Chow et al, 2005 (PMID: 16080188). In brief, human whole blood was fixed in 4% formaldehyde (methanol-free) for 10 min at 22°C. Red blood cells were then lyzed by the addition of Triton X-100 (final concentration - 0.1%) for 15 min at 37°C. For experimentation, cells were treated with 50% methanol (-20°C) for 15 min at 4°C. Cells were then incubated with the antibody (ab20181, 11.117µg/ml) for 30 min at 4°C. The secondary antibody used was Alexa Fluor® 488 goat anti-mouse IgG (H&L) (ab150113) at 1/2000 dilution for 30 min at 4°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >30,000 total events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. Gating strategy – peripheral blood lymphocytes.

Ab20181 staining human normal thymus tissue. Staining is localised to cellular membranes.
Left panel: with primary antibody at 0.334µg/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 antigen retrieval buffer EDTA pH 9.0 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 minutes 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.

Anti-HLA-DR antibody [TAL 1B5] (ab20181) at 0.667 µg/ml + whole tissue lysate prepared from human colon at 50 µg

Secondary
HRP conjugated goat anti-mouse polyclonal at 1/3000 dilution

Developed using the ECL technique.

Predicted band size: 29 kDa
Observed band size: 33 kDa why is the actual band size different from the predicted?


Exposure time: 5 seconds

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Human peripheral blood mononuclear cells isolated from whole blood were treated with BD Cytofix/Cytoperm™ kit for intracellular staining. Cells were then incubated in 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab20181, 11.117µg/ml) for 30 min at 4°C. The secondary antibody used was Alexa Fluor® 488 goat anti-mouse IgG (H&L) (ab150117) at 1/2000 dilution for 30 min at 4°C.

Isotype control antibody (black line) was mouse IgG1 [15-6E10A7] (ab170190, 0.005μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control.

Acquisition of 10,000 total events were collected using a 50mW Blue laser (488nm) and 530/30 bandpass filter. Gating strategy – lymphocytes.