IHC image of Islet 1 staining in a section of formalin fixed, paraffin embedded mouse embryo E12, performed on a Leica Bond™ system using the standard protocol B. The section was pre-treated using heat mediated antigen retrieval (EDTA based pH 9.0 solution, epitope retrieval solution 2) for 20 mins. The section was then incubated with ab20670, 0.5µg/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.

IHC image of Islet 1 staining in a section of formalin fixed, paraffin embedded mouse embryo E12, performed on a Leica Bond™ system using the standard protocol B. The section was pre-treated using heat mediated antigen retrieval (EDTA based pH 9.0 solution, epitope retrieval solution 2) for 20 mins. The section was then incubated with ab20670, 0.5µg/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.

ab20670 2µg/ml staining ISLET1 in human pancreas using an automated system (DAKO Autostainer Plus). Using this protocol there is strong nuclear and weak cytoplasmic staining primarily in the pancreatic islet.
Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer EDTA pH 9.0 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, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.

Dorsal root ganglion explants were dissected from 16 day-old rat embryos and cultured for 6 hours in vitro with Neurobasal Medium containing B27 supplement.     

Nuclei stained positive for anti-Islet 1 antibody ab20670 at 2µg/ml. As would be expected, not all cells in this preparation were Islet 1-positive. Pre-incubation of ab20670 with the immunizing peptide ab21996 resulted in complete blocking of the antibody.

Green = ab20670
Blue = To-pro-3 nuclear stain

The level of magnification is different in each image.    

Immunocytochemistry/ Immunofluorescence analysis of adult mouse cardiac fibroblasts labeling Islet 1 with ab20670 at 1/100 dilution (green). Samples were fixed using 4% PFA with 0.25% TritonX-100. Scale bar is 100 µM.

Immunohistochemistry (Frozen sections) analysis of mouse brain tissue sections labeling Islet 1 with ab20670 at 1/200 dilution. The tissue was fixed with paraformaldehyde followed by blocking with 5% serum for 1 hour at 25°C. The tissue was incubated with ab20670 in PBST for 12 hours at 4°C. A polyclonal donkey anti-rabbit Alexa Fluor^® 594 secondary antibody was used at 1/1000 dilution.

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Immunocytochemistry/ Immunofluorescence analysis of mouse dorsal root ganglia (DRG) neurons labeling Islet 1 with ab20670 at 1/200 dilution. Cells were fixed with paraformaldehyde and permeabilized with Tween 20. Blocking of the cells was performed with 2% serum for 30 minutes at 21°C, followed by incubation with ab20670 for 18 hours at 4°C. A polyclonal goat anti-rabbit Alexa Fluor^® 568 secondary antibody was used at 1/1000 dilution. DAPI was used to counterstain.

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Immunohistochemical analysis of paraffin-embedded mouse E11.5 somites labelling Islet 1 with ab20670 at 1/100 dilution, followed by BioGenex polymer-HRP reagent (undiluted according to manufacturer).

ab20670 detected Islet 1 in the nucleus of frozen sections of E11.5 mouse embryonic brain using 1 ug/ml of antibody. Brighter staining may be achieved using a greater concentration of antibody, e.g. 2.5 or 5 ug/ml.