We have had mixed results for use of this antibody in IHC-P using Mouse tissue samples. The abReview data below from external researchers reports good staining with this antibody in Mouse tissue samples using IHC-Fr.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
1/200. (See abreview 24266)
Use a concentration of 1 µg/ml. Detects a band of approximately 25 kDa (predicted molecular weight: 29 kDa).Can be blocked with Human Neutrophil Elastase peptide (ab68671). Abcam recommends using milk as the blocking agent. Abcam welcomes customer feedback and would appreciate any comments regarding this product and the data presented above.
1/100 - 1/250. PubMed: 22266908
1/2000. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
Modifies the functions of natural killer cells, monocytes and granulocytes. Inhibits C5a-dependent neutrophil enzyme release and chemotaxis.
Bone marrow cells.
Involvement in disease
Defects in ELANE are a cause of cyclic haematopoiesis (CH) [MIM:162800]; also known as cyclic neutropenia. CH is an autosomal dominant disease in which blood-cell production from the bone marrow oscillates with 21-day periodicity. Circulating neutrophils vary between almost normal numbers and zero. During intervals of neutropenia, affected individuals are at risk for opportunistic infection. Monocytes, platelets, lymphocytes and reticulocytes also cycle with the same frequency. Defects in ELANE are the cause of neutropenia severe congenital autosomal dominant type 1 (SCN1) [MIM:202700]. SCN1 is a disorder of hematopoiesis characterized by a maturation arrest of granulopoiesis at the level of promyelocytes with peripheral blood absolute neutrophil counts below 0.5 x 10(9)/l and early onset of severe bacterial infections.
Belongs to the peptidase S1 family. Elastase subfamily. Contains 1 peptidase S1 domain.
Immunocytochemistry/ Immunofluorescence - Anti-Neutrophil Elastase antibody (ab68672)This image is courtesy of an Abreview submitted by Ruma Raha-Chowdhury
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.
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
Western blot - Anti-Neutrophil Elastase antibody (ab68672)
Lanes 1-2 : Anti-Neutrophil Elastase antibody (ab68672) at 1 µg/ml Lanes 3-4 : Anti-Neutrophil Elastase antibody (ab68672) at 1 µg/ml (Milk blocking has been used)
Lanes 1 & 3 : Bone Marrow (Human) Tissue Lysate - adult normal tissue Lanes 2 & 4 : Mouse Bone Marrow Tissue Lysate
Lysates/proteins at 20 µg per lane.
Secondary All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution
Immunohistochemistry (Frozen sections) - Anti-Neutrophil Elastase antibody (ab68672)Image courtesy of Simon Cleary
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.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Neutrophil Elastase antibody (ab68672)This image is courtesy of an abreview submitted by Carl Hobbs, King's College London, United Kingdom
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).