Anti-Neutrophil Elastase antibody (ab68672)

Immunofluorescent staining with ab68672 of blood film of trangenic mouse that suffered from systemic inflamation. Neutrophil Elastase (in green) expressed in the lysosomes of activated neutrophils. Cells were fixed with methanol and permeabilized with 0.3% TritonX in 0.1% PBS. Blocking was performed with 10% serum for 15 minutes at 24ºC. This was followed by 1 hour incubation at 24ºC with ab68672 which was used at 1/200. The secondary antibody used was an anti-rabbit Alexa Fluor 488.

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IHC image of Neutrophil Elastase staining in human spleen 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 ab68672, 1µg/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

This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 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 ab68672 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.

ab68672 staining Neutrophil Elastase in Mouse LPS treated Lung tissue sections by Immunohistochemistry (IHC-Fr - frozen sections). Tissue was fixed with formaldehyde and blocked with 1% BSA for 10 minutes. Samples were incubated with primary antibody (1/50) for 2 hours. A Biotin-conjugated Goat anti-rabbit IgG polyclonal (1/200) was used as the secondary antibody.

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Immunohistochemistical detection of Neutrophil Elastase using antibody (ab68672) on formaldehyde-fixed paraffin-embedded human spleen sections. Antigen retrieval step: heat mediated in citric acid pH6 buffer. Permeabilization: No Blocking step: 1% BSA for 10 mins @ rt°C. Primary antibody dilution 1/2000; incubation time: 2 hours in TBS/BSA/azide. Secondary Antibody: anti Rabbit IgG conjugated to biotin (1/200).

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