Immunofluorescence (IF)
(Image: FOX3 (red) and and beta-tubulin (Green) staining of cochlear ganglion of a neonatal mouse. )
Chicken anti-Human, Mouse Neu-N (Fox 3) Antibody | anti-NeuN antibody
Neu-N (Fox 3), Affinity Purified Antibody, Chicken
Reactivity
Human, Mouse
Applications
Immunohistochemistry
Purity
Affinity Purified
Synonyms
Neu-N (Fox 3); Affinity Purified Antibody; Chicken; anti-NeuN antibody
Host
Chicken
Reactivity
Human, Mouse
Purity/Purification
Affinity Purified
Form/Format
Liquid PBS (pH 7.2; 10 mM; isotonic 0.9%, w/v) with sodium azide (0.02%, w/v).
Concentration
100 ug/ml (varies by lot)
Applicable Applications for anti-NeuN antibody
Immunohistochemistry (IHC)
Application Notes
Immunohistochemistry: 1:1000
Dilutions listed as a recommendation. Optimal dilution should be determined by investigator.
Immunostaining Cell Cultures:
1. Draw of culture medium with aspirator and add 1 ml of 3.7 % formalin in PBS solution to the dish. (make up from 10mls Fisher 37% formalin plus 90mls PBS, the Fisher formalin contains 37% formaldehyde plus about 1% methanol which may be relevant sometimes). Let sit at room temp for 1 minute. (can add 0.1% Tween 20 to PBS used here and all subsequent steps to reduce background; probably best not to do this first time round though as it may extract your antigen or help wash your cells off the dish).
2. Take off the formalin/PBS and add 1ml of cold methanol (-20°C, kept in well sealed bottle in fridge). Let sit for no more than 1 minute.
3. Take off methanol and add 1ml of PBS, not letting the specimen dry out. To block nonspecific antibody binding can add ~10ml (=1%) of goat serum (Sigma), and can incubate for 30 minutes. Can then add antibody reagents. Typically 100ml of hybridoma tissue culture supernatent or 1ml of mouse ascites fluid or crude serum. Incubate for 1 hour at room temp. (or can go at 37°C for 30 minutes to 1 hour, or can do 4°C overnight, exact time not too critical). Can do very gentle shaking for well adherent cell lines (3T3, Hek293 etc.).
4. Remove primary antibody and replace with 1 ml of PBS. Let sit for 5-10 minutes, replace PBS and repeat twice, to give three washes in PBS.
5. Add 0.5 mls of secondary antibody. These are fluorescently labeled Goat anti mouse or rabbit antibodies and are conjugated to ALEXA dyes and are from Molecular probes (Eugene Oregon, the ALEXA dyes are sulphonated rhodamine compounds and are much more stable to UV than FITC, TRITC, Texas red etc.). Typically make 1:2,000 dilutions of these secondaries in PBS plus 1% goat serum, BSA or non fat milk carrier. Incubate for 1 hour at room temp. (or can go at 37°C for 30 minutes to 1 hour, or can do 4°C overnight). Can do gentle shaking for well adherent cell lines (3T3, HEK293 etc.).
6. Remove secondary antibody and replace with 1 ml of PBS. Let sit for 5-10 minutes, replace PBS and repeat twice, to give three washes in PBS.
7. Drop on one drop of Fisher mounting medium onto dish and apply 22mm square coverslip. View in the microscope!
Immunostaining Tissue:
Solutions
PBS - sodium phosphate-buffered (100 mM; pH 7.2) isotonic (0.9% NaCl, w/v) saline Antibody dilution buffer (PBS with 0.1% non-ionic detergent, such as Triton X-100 or Tween-20). For anti-fading, make your own fluorescein anti-fading reagent -- Make up a 2 mg/ml phenylene diamine solution in PBS (phenylene diamine requires extensive vortexing to put it into solution). Once the phenylene diamine is completely dissolved, add an equal volume of glycerol and mix. This reagent will last about a week at -20OC. Discard this reagent when it starts to turn dark brown.
Other Reagents
Fluorescein-labeled goat anti-chicken IgY
1. Prepare your tissue sections or cultured cells as you normally would. Wash your sections or cells for 1 min with PBS at room temperature.
2. Incubate your sections or cells with your chicken primary antibodies (diluted in "antibody dilution buffer") for at least 1 hour at room temperature. The concentration of your antibody may be anywhere from 1:50-1:150 depending on the titre of the antibody and the concentration of your antigen.
3. Wash your sections or cells over a 10 minute period at room temperature (with two changes of PBS).
4. Incubate your sections or cells with fluorescein-labeled goat anti-chicken IgY (1:500 dilution in "antibody dilution buffer” for 1 hour at room temperature. Be sure to keep these slides or culture dishes in subdued light (e.g., in a drawer) to avoid bleaching of the fluorescein dye.
5. Repeat step #4
6. Add a drop of "fluorescence anti-fading reagent" to your sections or cells. Place a coverslip over the section. If you want to reduce messiness, you may also seal the coverslip by painting the edges with nail polish.
7. Store the slides or culture dishes in the refrigerator (in the dark).
Dilutions listed as a recommendation. Optimal dilution should be determined by investigator.
Immunostaining Cell Cultures:
1. Draw of culture medium with aspirator and add 1 ml of 3.7 % formalin in PBS solution to the dish. (make up from 10mls Fisher 37% formalin plus 90mls PBS, the Fisher formalin contains 37% formaldehyde plus about 1% methanol which may be relevant sometimes). Let sit at room temp for 1 minute. (can add 0.1% Tween 20 to PBS used here and all subsequent steps to reduce background; probably best not to do this first time round though as it may extract your antigen or help wash your cells off the dish).
2. Take off the formalin/PBS and add 1ml of cold methanol (-20°C, kept in well sealed bottle in fridge). Let sit for no more than 1 minute.
3. Take off methanol and add 1ml of PBS, not letting the specimen dry out. To block nonspecific antibody binding can add ~10ml (=1%) of goat serum (Sigma), and can incubate for 30 minutes. Can then add antibody reagents. Typically 100ml of hybridoma tissue culture supernatent or 1ml of mouse ascites fluid or crude serum. Incubate for 1 hour at room temp. (or can go at 37°C for 30 minutes to 1 hour, or can do 4°C overnight, exact time not too critical). Can do very gentle shaking for well adherent cell lines (3T3, Hek293 etc.).
4. Remove primary antibody and replace with 1 ml of PBS. Let sit for 5-10 minutes, replace PBS and repeat twice, to give three washes in PBS.
5. Add 0.5 mls of secondary antibody. These are fluorescently labeled Goat anti mouse or rabbit antibodies and are conjugated to ALEXA dyes and are from Molecular probes (Eugene Oregon, the ALEXA dyes are sulphonated rhodamine compounds and are much more stable to UV than FITC, TRITC, Texas red etc.). Typically make 1:2,000 dilutions of these secondaries in PBS plus 1% goat serum, BSA or non fat milk carrier. Incubate for 1 hour at room temp. (or can go at 37°C for 30 minutes to 1 hour, or can do 4°C overnight). Can do gentle shaking for well adherent cell lines (3T3, HEK293 etc.).
6. Remove secondary antibody and replace with 1 ml of PBS. Let sit for 5-10 minutes, replace PBS and repeat twice, to give three washes in PBS.
7. Drop on one drop of Fisher mounting medium onto dish and apply 22mm square coverslip. View in the microscope!
Immunostaining Tissue:
Solutions
PBS - sodium phosphate-buffered (100 mM; pH 7.2) isotonic (0.9% NaCl, w/v) saline Antibody dilution buffer (PBS with 0.1% non-ionic detergent, such as Triton X-100 or Tween-20). For anti-fading, make your own fluorescein anti-fading reagent -- Make up a 2 mg/ml phenylene diamine solution in PBS (phenylene diamine requires extensive vortexing to put it into solution). Once the phenylene diamine is completely dissolved, add an equal volume of glycerol and mix. This reagent will last about a week at -20OC. Discard this reagent when it starts to turn dark brown.
Other Reagents
Fluorescein-labeled goat anti-chicken IgY
1. Prepare your tissue sections or cultured cells as you normally would. Wash your sections or cells for 1 min with PBS at room temperature.
2. Incubate your sections or cells with your chicken primary antibodies (diluted in "antibody dilution buffer") for at least 1 hour at room temperature. The concentration of your antibody may be anywhere from 1:50-1:150 depending on the titre of the antibody and the concentration of your antigen.
3. Wash your sections or cells over a 10 minute period at room temperature (with two changes of PBS).
4. Incubate your sections or cells with fluorescein-labeled goat anti-chicken IgY (1:500 dilution in "antibody dilution buffer” for 1 hour at room temperature. Be sure to keep these slides or culture dishes in subdued light (e.g., in a drawer) to avoid bleaching of the fluorescein dye.
5. Repeat step #4
6. Add a drop of "fluorescence anti-fading reagent" to your sections or cells. Place a coverslip over the section. If you want to reduce messiness, you may also seal the coverslip by painting the edges with nail polish.
7. Store the slides or culture dishes in the refrigerator (in the dark).
Immunogen Sequence
Synthetic peptide-keyhole limpet hemocyanin (KLH) conjugate. This synthetic peptide corresponded to the Fox-3 gene product (also known as the Neu-N antigen), but was shared between the human NP_001076044, NCBI) and mouse (NP_001020102, NCBI) sequences.
Preparation and Storage
Store at 4°C in the dark. Under these conditions , the antibody should have a shelf life of at least 12 months (provided they remain sterile). Do not freeze the antibody unless you want to store it for longer periods of time. Note, however, that each time an antibody preperation is frozen, about half of its binding activity is lost.
Immunofluorescence (IF)
(Image: FOX3 (red) and and beta-tubulin (Green) staining of cochlear ganglion of a neonatal mouse. )
Immunofluorescence (IF)
(Image: FOX3 (red) and and beta-tubulin (Green) staining of cochlear ganglion of a neonatal mouse. )
Product Categories/Family for anti-NeuN antibody
Similar Products
Product Notes
The NeuN (Catalog #AAA555371) is an Antibody produced from Chicken and is intended for research purposes only. The product is available for immediate purchase. The Neu-N (Fox 3), Affinity Purified Antibody, Chicken reacts with Human, Mouse and may cross-react with other species as described in the data sheet. AAA Biotech's Neu-N (Fox 3) can be used in a range of immunoassay formats including, but not limited to, Immunohistochemistry (IHC). Immunohistochemistry: 1:1000 Dilutions listed as a recommendation. Optimal dilution should be determined by investigator. Immunostaining Cell Cultures: 1. Draw of culture medium with aspirator and add 1 ml of 3.7 % formalin in PBS solution to the dish. (make up from 10mls Fisher 37% formalin plus 90mls PBS, the Fisher formalin contains 37% formaldehyde plus about 1% methanol which may be relevant sometimes). Let sit at room temp for 1 minute. (can add 0.1% Tween 20 to PBS used here and all subsequent steps to reduce background; probably best not to do this first time round though as it may extract your antigen or help wash your cells off the dish). 2. Take off the formalin/PBS and add 1ml of cold methanol (-20°C, kept in well sealed bottle in fridge). Let sit for no more than 1 minute. 3. Take off methanol and add 1ml of PBS, not letting the specimen dry out. To block nonspecific antibody binding can add ~10ml (=1%) of goat serum (Sigma), and can incubate for 30 minutes. Can then add antibody reagents. Typically 100ml of hybridoma tissue culture supernatent or 1ml of mouse ascites fluid or crude serum. Incubate for 1 hour at room temp. (or can go at 37°C for 30 minutes to 1 hour, or can do 4°C overnight, exact time not too critical). Can do very gentle shaking for well adherent cell lines (3T3, Hek293 etc.). 4. Remove primary antibody and replace with 1 ml of PBS. Let sit for 5-10 minutes, replace PBS and repeat twice, to give three washes in PBS. 5. Add 0.5 mls of secondary antibody. These are fluorescently labeled Goat anti mouse or rabbit antibodies and are conjugated to ALEXA dyes and are from Molecular probes (Eugene Oregon, the ALEXA dyes are sulphonated rhodamine compounds and are much more stable to UV than FITC, TRITC, Texas red etc.). Typically make 1:2,000 dilutions of these secondaries in PBS plus 1% goat serum, BSA or non fat milk carrier. Incubate for 1 hour at room temp. (or can go at 37°C for 30 minutes to 1 hour, or can do 4°C overnight). Can do gentle shaking for well adherent cell lines (3T3, HEK293 etc.). 6. Remove secondary antibody and replace with 1 ml of PBS. Let sit for 5-10 minutes, replace PBS and repeat twice, to give three washes in PBS. 7. Drop on one drop of Fisher mounting medium onto dish and apply 22mm square coverslip. View in the microscope! Immunostaining Tissue: Solutions PBS - sodium phosphate-buffered (100 mM; pH 7.2) isotonic (0.9% NaCl, w/v) saline Antibody dilution buffer (PBS with 0.1% non-ionic detergent, such as Triton X-100 or Tween-20). For anti-fading, make your own fluorescein anti-fading reagent -- Make up a 2 mg/ml phenylene diamine solution in PBS (phenylene diamine requires extensive vortexing to put it into solution). Once the phenylene diamine is completely dissolved, add an equal volume of glycerol and mix. This reagent will last about a week at -20OC. Discard this reagent when it starts to turn dark brown. Other Reagents Fluorescein-labeled goat anti-chicken IgY 1. Prepare your tissue sections or cultured cells as you normally would. Wash your sections or cells for 1 min with PBS at room temperature. 2. Incubate your sections or cells with your chicken primary antibodies (diluted in "antibody dilution buffer") for at least 1 hour at room temperature. The concentration of your antibody may be anywhere from 1:50-1:150 depending on the titre of the antibody and the concentration of your antigen. 3. Wash your sections or cells over a 10 minute period at room temperature (with two changes of PBS). 4. Incubate your sections or cells with fluorescein-labeled goat anti-chicken IgY (1:500 dilution in "antibody dilution buffer” for 1 hour at room temperature. Be sure to keep these slides or culture dishes in subdued light (e.g., in a drawer) to avoid bleaching of the fluorescein dye. 5. Repeat step #4 6. Add a drop of "fluorescence anti-fading reagent" to your sections or cells. Place a coverslip over the section. If you want to reduce messiness, you may also seal the coverslip by painting the edges with nail polish. 7. Store the slides or culture dishes in the refrigerator (in the dark). Researchers should empirically determine the suitability of the NeuN for an application not listed in the data sheet. Researchers commonly develop new applications and it is an integral, important part of the investigative research process. It is sometimes possible for the material contained within the vial of "Neu-N (Fox 3), Antibody" to become dispersed throughout the inside of the vial, particularly around the seal of said vial, during shipment and storage. We always suggest centrifuging these vials to consolidate all of the liquid away from the lid and to the bottom of the vial prior to opening. Please be advised that certain products may require dry ice for shipping and that, if this is the case, an additional dry ice fee may also be required.Precautions
All products in the AAA Biotech catalog are strictly for research-use only, and are absolutely not suitable for use in any sort of medical, therapeutic, prophylactic, in-vivo, or diagnostic capacity. By purchasing a product from AAA Biotech, you are explicitly certifying that said products will be properly tested and used in line with industry standard. AAA Biotech and its authorized distribution partners reserve the right to refuse to fulfill any order if we have any indication that a purchaser may be intending to use a product outside of our accepted criteria.Disclaimer
Though we do strive to guarantee the information represented in this datasheet, AAA Biotech cannot be held responsible for any oversights or imprecisions. AAA Biotech reserves the right to adjust any aspect of this datasheet at any time and without notice. It is the responsibility of the customer to inform AAA Biotech of any product performance issues observed or experienced within 30 days of receipt of said product. To see additional details on this or any of our other policies, please see our Terms & Conditions page.Item has been added to Shopping Cart
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