IHC image of Nestin staining in a section of formalin-fixed paraffin-embedded normal rat brain performed on a Leica BOND^TM 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 20mins. The section was then incubated with ab6142, 0.05ug/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 hematoxylin 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-Nestin antibody [Rat-401] - Neural Stem Cell Marker (ab6142) at 1 µg/ml

Lane 1 : Mouse E12 brain tissue lysate
Lane 2 : Mouse E14 brain tissue lysate
Lane 3 : Mouse E16 brain tissue lysate
Lane 4 : Mouse E18 brain tissue lysate
Lane 5 : Rat E12 brain tissue lysate
Lane 6 : Rat E14 brain tissue lysate
Lane 7 : Rat E16 brain tissue lysate
Lane 8 : Rat E18 brain tissue lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 200 kDa

This blot was produced using a 3-8% Tris-Acetate gel under the Tris-Acetate buffer system. The gel was run at 150V for 60 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% milk before being incubated with ab6142 overnight at 4°C. Antibody binding was detected using Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) at 1/20000 dilution for 1 hour at room temperature before imaging.

REST expression in N-hREST mouse brains correlates with stemness in embryonic neural stem cells. Immunofluorescence analysis of E18.5 N-hREST and LSL-hRESTcontrol littermate mouse brains with antibodies against REST (using an antibody that preferentially recognizes hREST over mouse REST) and Nestin (using ab6142)

Mice were anesthetized and perfused with phosphate-buffered saline followed by 4% paraformaldehyde (PFA). Brain tissues were then dissected and fixed in 4% PFA overnight at 4 °C. Fixed brain tissues were processed for paraffin embedding and then cut into 5-μm sections. 

ab6142 staining adult mouse brain tissue section by Immunohistochemistry (Formalin/PFA-fixed, paraffin embedded sections). Tissue underwent fixation in paraformaldehyde, heat mediated antigen retrieval in Sodium Citrate, permeabilization in 1% Triton buffer and blocking in 10% serum for 1 hour at 25°C. The primary antibody, diluted 1/200 (PBS, 2% Donkey serum, 0.2% Triton) for 16 hours at 4°C.  An Alexa Fluor® 488 conjugated donkey polyclonal to mouse Ig, diluted 1/500 was used as the secondary.

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