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Our Abpromise guarantee covers the use of ab7751 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IHC-P||Use a concentration of 10 µg/ml. Pretreatment: 0.1% pepsin (trypsin) in 0.1 M HCl for 30 minutes at RT. High-temperature citrate buffer antigen retrieval can also be performed.|
|WB||Use a concentration of 1 - 2 µg/ml. We suggest reducing conditions and a 90 minute incubation time.|
|IHC-FoFr||Use at an assay dependent concentration.|
|IHC-Fr||Use at an assay dependent concentration.|
|IHC-P||Use a concentration of 5 µg/ml.|
|IP||Use at an assay dependent concentration.|
|Flow Cyt||1/20 - 1/50.
ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.
This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using LI-COR® blocking buffer before being incubated with ab7751 overnight at 4°C. Antibody binding was detected using the IRDye® 800CW Goat Anti-Mouse secondary at a 1:10,000 dilution for 1hr at room temperature and then imaged using the Odyssey® CLx Imaging System.
IHC image of ab7751 staining beta III Tubulin in human cerebellum formalin fixed paraffin embedded tissue sections, performed on a Leica Bond. 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 ab7751, 5μg/ml dilution, 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. No primary antibody was used in the secondary only control (shown on the inset).
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.
ab7751 staining beta III Tubulin in induced Neuron derived from Human pluripotent cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with 0.25% Triton X-100 in PBS and blocked with 1% BSA for 30 minutes at 22°C. Samples were incubated with primary antibody (1/500) for 16 hours at 4°C. An Alexa Fluor® 594-conjugated Donkey anti-mouse IgG polyclonal (1/1000) was used as the secondary antibody.
ab7751 staining Neuron specific beta III Tubulin in human colon tissue sections by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue underwent formaldehyde fixation before heat mediated antigen retrieval in Citric acid pH6 and then blocking with 1% BSA was performed for 10 minutes at RT. The primary antibody was used at dilution at 1/200 and incubated with sample in TBS/BSA/azide for 2 hours. A Biotin conjugated goat polyclonal to mouse IgG was used at dilution at 1/200 as secondary antibody. Images A and B represent staining of the clone TU20 and 2G10 respectively in ganglia of Auerbach's plexus.
ab7751 staining beta III Tubulin in Neuro-2a cells. The cells were fixed with 100% methanol (5min) and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated with ab7751 at 1/1000 and ab6046 at 1µg/ml overnight at +4°C, followed by a further incubation at room temperature for 1h with an AlexaFluor®488 Goat anti-Mouse secondary (ab150117) at 2 μg/ml (shown in green) and AlexaFluor®594 Goat anti-Rabbit secondary (ab150088) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labelled in blue with DAPI.
Negative controls: 1– Rabbit primary and anti-mouse secondary antibody; 2 – Mouse primary antibody and anti-rabbit secondary antibody. Controls 1 and 2 indicate that there is no unspecific reaction between primary and secondary antibodies used.
Immunohistochemistical detection of Neuron specific beta III Tubulin using antibody (ab7751) on whole mouse e12 embryo (formaldehyde fixed/frozen sections). Permeabilization: No. Blocking step: 1% BSA for 10 mins @ rt°C. Primary Antibody Dilution 1/200; Incubation time 2 hours in TBS/BSA/azide. Secondary antibody: anti Mouse IgG Conjugated to Alexa Fluor® 594 (1/1000). Floorplate region of developing cervical spinal cord (I have not studied this particular region).
ab7751 immunostaining (1/500) neuronal processes in primary mouse cortical culture.
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