IHC image of NeuroD2 staining in a formalin fixed, paraffin embedded mouse brain 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 ab104430, 5 µg/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-NeuroD2 antibody (ab104430) at 1 µg/ml

Lane 1 : Human brain tissue lysate - total protein (ab29466)
Lane 2 : Cerebellum Mouse Tissue Lysate
Lane 3 : Mouse Hippocampus Tissue Lysate
Lane 4 : P0 Mouse Brain Mouse Tissue Lysate
Lane 5 : P7 Mouse Brain Tissue Lysate
Lane 6 : Human brain tissue lysate - total protein (ab29466) with Immunising peptide at 1 µg/ml
Lane 7 : Cerebellum Mouse Tissue Lysate with Immunising peptide at 1 µg/ml
Lane 8 : Mouse Hippocampus Tissue Lysate with Immunising peptide at 1 µg/ml
Lane 9 : P0 Mouse Brain Mouse Tissue Lysate with Immunising peptide at 1 µg/ml
Lane 10 : P7 Mouse Brain Tissue Lysate with Immunising peptide at 1 µg/ml

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 41 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?
Additional bands at: 21 kDa, 41 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 4 minutes

IHC-P image of NeuroD2 staining on mouse CA1 hippocampus sections using ab104430 (1:2000). The sections were deparaffinized and subjected to heat mediated antigen retrieval using citric acid. The sections wer then blocked using 1% BSA at 21°C for 10 min. The primary antibody was incubated at 21°C for 2 hours.

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IHC-P image of NeuroD2 staining on rat CA1 hippocampus sections using ab104430 (1:1000). The sections were deparaffinized and subjected to heat mediated antigen retrieval using citric acid. The sections wer then blocked using 1% BSA at 21°C for 10 min. The primary antibody was incubated at 21°C for 2 hours

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ab104430 staining NeuroD2 in 10% formaldehyde perfusion fixed 6 week old frozen mouse brain section (dentate gyrus). No antigen retrieval was performed. The section was incubated in 1% BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab104430, 1/2000 dilution) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. DAPI was used to stain the cell nuclei (blue).