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RecombinantRabMAb

Recombinant Anti-NAPSIN A antibody [EPR6257] (ab129189)

  • Datasheet
  • SDS
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NAPSIN A antibody [EPR6257] (ab129189)
  • Western blot - Anti-NAPSIN A antibody [EPR6257] (ab129189)
  • Western blot - Anti-NAPSIN A antibody [EPR6257] (ab129189)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NAPSIN A antibody [EPR6257] (ab129189)
  • OI-RD Scanning - Anti-NAPSIN A antibody [EPR6257] (ab129189)
  • Anti-NAPSIN A antibody [EPR6257] (ab129189)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR6257] to NAPSIN A
  • Suitable for: WB, IHC-P
  • Reacts with: Human

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Overview

  • Product name

    Anti-NAPSIN A antibody [EPR6257]
    See all NAPSIN A primary antibodies
  • Description

    Rabbit monoclonal [EPR6257] to NAPSIN A
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WB, IHC-Pmore details
    Unsuitable for: ICC/IF
  • Species reactivity

    Reacts with: Human
  • Immunogen

    Synthetic peptide within Human NAPSIN A aa 50-150. The exact sequence is proprietary.

  • Positive control

    • WB: Human lung adenocarcinoma lysate. IHC-P: Human lung tissue.
  • General notes

    Mouse, Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    Reproducibility is key to advancing scientific discovery and accelerating scientists’ next breakthrough.

    Abcam is leading the way with our range of recombinant antibodies, knockout-validated antibodies and knockout cell lines, all of which support improved reproducibility.

    We are also planning to innovate the way in which we present recommended applications and species on our product datasheets, so that only applications & species that have been tested in our own labs, our suppliers or by selected trusted collaborators are covered by our Abpromise™ guarantee.

    In preparation for this, we have started to update the applications & species that this product is Abpromise guaranteed for.

    We are also updating the applications & species that this product has been “predicted to work with,” however this information is not covered by our Abpromise guarantee.

    Applications & species from publications and Abreviews that have not been tested in our own labs or in those of our suppliers are not covered by the Abpromise guarantee.

    Please check that this product meets your needs before purchasing. If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, as well as customer reviews and Q&As.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Stable for 12 months at -20°C.
  • Dissociation constant (KD)

    KD = 6.17 x 10 -11 M
    Learn more about KD
  • Storage buffer

    pH: 7.20
    Preservative: 0.01% Sodium azide
    Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    EPR6257
  • Isotype

    IgG
  • Research areas

    • Tags & Cell Markers
    • Cell Type Markers
    • Tumor Associated
    • Cell Biology
    • Proteolysis / Ubiquitin
    • Proteolytic enzymes
    • Other proteases

Associated products

  • Alternative Versions

    • Anti-NAPSIN A antibody [EPR6257] - BSA and Azide free (ab248340)
  • Compatible Secondaries

    • Goat Anti-Rabbit IgG H&L (HRP) (ab97051)
  • Isotype control

    • Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
  • Recombinant Protein

    • Recombinant Human NAPSIN A protein (denatured) (ab202144)

Applications

Our Abpromise guarantee covers the use of ab129189 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB 1/1000 - 1/10000. Detects a band of approximately 50 kDa (predicted molecular weight: 45 kDa).
IHC-P 1/100 - 1/500. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

  • Application notes
    Is unsuitable for ICC/IF.
  • Target

    • Function

      May be involved in processing of pneumocyte surfactant precursors.
    • Tissue specificity

      Expressed predominantly in adult lung (type II pneumocytes) and kidney and in fetal lung. Low levels in adult spleen and very low levels in peripheral blood leukocytes.
    • Sequence similarities

      Belongs to the peptidase A1 family.
    • Cellular localization

      Secreted.
    • Target information above from: UniProt accession O96009 The UniProt Consortium
      The Universal Protein Resource (UniProt) in 2010
      Nucleic Acids Res. 38:D142-D148 (2010) .

      Information by UniProt
    • Database links

      • Entrez Gene: 9476 Human
      • Omim: 605631 Human
      • SwissProt: O96009 Human
      • Unigene: 512843 Human
      • Alternative names

        • Asp 4 antibody
        • ASP4 antibody
        • Aspartyl protease 4 antibody
        • KAP antibody
        • Kdap antibody
        • Kidney derived aspartic protease like protein antibody
        • NAP1 antibody
        • NAPA antibody
        • Napsa antibody
        • NAPSA_HUMAN antibody
        • Napsin 1 antibody
        • napsin A aspartic peptidase antibody
        • Napsin A precursor antibody
        • Napsin-1 antibody
        • Napsin-A antibody
        • Pronapsin A antibody
        • SNAPA antibody
        • TA01/TA02 antibody
        see all

      Images

      • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NAPSIN A antibody [EPR6257] (ab129189)
        Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NAPSIN A antibody [EPR6257] (ab129189)

        Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human lung tissue labelling NAPSIN A with purified ab129189 at a dilution of 1/500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

      • Western blot - Anti-NAPSIN A antibody [EPR6257] (ab129189)
        Western blot - Anti-NAPSIN A antibody [EPR6257] (ab129189)
        Anti-NAPSIN A antibody [EPR6257] (ab129189) at 1/5000 dilution (purified) + Human lung adenocarcinoma tissue lysate at 20 µg

        Secondary
        HRP-conjugated goat anti-rabbit IgG (specific to the non-reduced form of IgG) at 1/1000 dilution

        Predicted band size: 45 kDa
        Observed band size: 50 kDa
        why is the actual band size different from the predicted?



        Blocking and dilution buffer: 5% NFDM /TBST.

      • Western blot - Anti-NAPSIN A antibody [EPR6257] (ab129189)
        Western blot - Anti-NAPSIN A antibody [EPR6257] (ab129189)
        Anti-NAPSIN A antibody [EPR6257] (ab129189) at 1/1000 dilution (unpurified) + Human lung adenocarcinoma lysate at 10 µg

        Secondary
        HRP-conjugated goat anti-rabbit IgG at 1/2000 dilution

        Predicted band size: 45 kDa

      • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NAPSIN A antibody [EPR6257] (ab129189)
        Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NAPSIN A antibody [EPR6257] (ab129189)

        Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human lung tissue labelling NAPSIN A with unpurified ab129189 at a dilution of 1/100.

        Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

      • OI-RD Scanning - Anti-NAPSIN A antibody [EPR6257] (ab129189)
        OI-RD Scanning - Anti-NAPSIN A antibody [EPR6257] (ab129189)
        Equilibrium disassociation constant (KD)
        Learn more about KD

        Click here to learn more about KD
      • Anti-NAPSIN A antibody [EPR6257] (ab129189)
        Anti-NAPSIN A antibody [EPR6257] (ab129189)

      Protocols

      • Western blot protocols
      • Immunohistochemistry protocols

      Click here to view the general protocols

      Datasheets and documents

      • Datasheet
      • SDS
    • References (0)

      Publishing research using ab129189? Please let us know so that we can cite the reference in this datasheet.

      ab129189 has not yet been referenced specifically in any publications.

      Customer reviews and Q&As

      Show All Reviews Q&A
      Submit a review Submit a question

      1-3 of 3 Abreviews or Q&A

      Question

      I kindly ask you to send me the protocols, information regarding pre-treatment etc. for the following abs:

      Read More

      Abcam community

      Verified customer

      Asked on Jun 21 2013

      Answer

      Our antigen retrieval method used for ab92378 and ab129189 is described below.

      Antigen Retrieval

      This is recommended Heat Induced Epitope Retrieval (HIER) using Decloaking Chamber/Pressure Cooker. Hot water bath or Microwave with temperature sensor can be also used (protocol would vary depending on the method used).

      1. Add 500 ml of dH 2O to Decloaker/Pressure Cooker.

      2. Immerse slides into staining dish containing Antigen Retrieval Solution. Place staining dish into decloaking chamber.

      3. Program to run for 30 seconds at 125° C, followed by 10 seconds at 90° C.

      4. Let it cool down to room temperature (10 - 20 minutes).

      5. Removes slides and rinse in TBST.

      6. Proceed to Staining step.

      Read More

      Tanya Bagrij

      Abcam Scientific Support

      Answered on Jun 21 2013

      Question

      In the specification sheet for Pax8 on your website (see in attachment) stands 100 ug and in the mail below – 250 ul. Can you please clarify this to me?

      Our customer is asking for this antibodies for IHC. Can you please send me the working procedure for the antibodies.

      Thank you in advance.

      Best regards,

      Read More

      Abcam community

      Verified customer

      Asked on Sep 27 2012

      Answer

      Thank you fo ryour message.

      To clarify regarding ab135618 Anti-PAX8 antibody, the datasheet states we sell this at 100 ug, at a concentration of 0.25 mg/ml. Therefore, you can calculate that at this concentrations, 100 ug will be in 250 ul of liquid.

      For the protocols, I am pleased to provide the general testing IHC-P protocols from the originators. I have copied thesebelow. I am sorry it has taken a while to obtain these.Please note that these will be a guideline only and further individual optimizationmay be required.

      As stated in the previous email, recommended dilutions in IHC-P for these antibodies are listed on the datasheets:

      ab129189 NAPSIN AIHC-P: 1/100 - 1/250.
      ab92378 HNF4 IHC-P: 1/100 - 1/250
      ab135618 PAX8IHC-P: 1/50 - 1/100.

      I hope this will be helpful to you. If you have any further questions, please do not hesitate to contact us.

      Protocol for ab135618 Anti-PAX8 antibody


      Preparing Tissue Sections for Immunostaining:

      1. Fix the tissue in 10% formalin at 4oC overnight.
      2. Embed fixed tissue in paraffin.
      3. Mount tissue sections on slides.
      4. Clear the paraffin with xylene for ten minutes; move slides to a fresh dish of xylene for an additional ten minutes. NOTE: Perform all xylene washes in a fume hood!
      5. Rinse the slides twice for 2 minutes in 100% alcohols (18:1:1 100% ethanol: 100% methanol: 100% isopropanol).
      6. Rinse the slides twice for 2 minutes in a 95% solution of the 100% alcohols.
      7. Place slides in an 80% solution of the 100% alcohols for 2 minutes, followed by deionized water for 5 minutes.
      8. Rinse slides several times with fresh deionized water followed by another five minutes wash using fresh water.


      Sodium Citrate Antigen Retrieval:

      1. Place slides in a glass slide holder and fill in the rest of the rack with blank slides (10 totals) to ensure even heating.
      2. Place rack in 600 ml of 10 mM sodium citrate, pH 6.0 in a glass 2 L-beaker. Mark a line at the top of the liquid on the beaker.
      3. Microwave for 20 min total, replacing evaporated water every 5 min.
      4. Cool slides for 20 min.
      5. Wash 4 X 3 min in ddH2O, and 3 min in 1X PBS.


      Blocking


      1. Block endogenous peroxidases by soaking slides in a solution of 90% methanol/3% H2O2 for 15 minutes at room temperature. Wash 3 X in PBS.
      2. Immerse slides in a dish containing blocking buffer (serum from host species of secondary antibody to be used, diluted 1:10 in TBS). Incubate at 37oC for one hour.


      Incubation with Primary Antibodies


      1. Cover the tissue sections with primary antibody diluted in blocking buffer. Antibody is diluted 1:50 and 1:100. Incubate for 1 hour at 37oC.
      2. Blot excess liquid from slides and rinse three times in PBS for five minutes each wash.
      Incubation with Secondary Antibodies
      3. Cover the tissue sections with secondary antibody diluted in blocking buffer according to manufacturer’s instructions. We routinely use prediluted universal secondary antibody (Jackson ImmunoResearch Laboratories). Incubate at 37oC for 30 min.
      4. Blot excess liquid and rinse twice in TBS for five minutes each wash.


      Counterstaining and Visualization

      1. Counterstain with Hematoxylin.
      2. Rinse several times in deionized water. Blot excess water around tissue, then apply one drop of mounting media to tissue and place coverslip over slide. Seal with nail polish.

      Citrate Solutions:
      Description: Formalin or other aldehyde fixation forms protein cross-links that mask the antigenic sites in tissue specimens, thereby giving weak or false negative staining for immunohistochemical detection of certain proteins. The citrate based solution is designed to break the protein cross-links, thereby unmasking the antigens and epitopes in formalin-fixed and paraffin embedded tissue sections and enhancing the staining intensity of antibodies.


      Sodium Citrate Buffer (10mM Sodium Citrate, 0.05% Tween 20, pH 6.0):

      Tri-sodium citrate (dihydrate) 2.94 g
      Distilled water 1000 ml
      Mix to dissolve. Adjust pH to 6.0 with 1N HCl and then add 0.5 ml of Tween 20 and mix well. Store this solution at room temperature for 3 months or at 4¢XC for longer storage.
      Citrate Buffer (10mM Citric Acid, 0.05% Tween 20, pH 6.0):
      Citric acid (anhydrous) 1.92 g
      Distilled water 1000 ml
      Mix to dissolve. Adjust pH to 6.0 with 1N NaOH and then add 0.5 ml of Tween 20 and mix well. Store this solution at room temperature for 3 months or at 4oC for longer storage.

      Washing Buffer:

      1 X PBS:
      NaCl 8 g
      KCl 0.2 g
      Na2HPO 41.44 g
      KH2PO4 0.24 g
      Distilled Water 800 mL
      Adjust pH to 7.2 with HCl.
      Adjust volume to 1 L with additional H2O


      Normal Serum Blocking Buffer:
      2% serum from host species of secondary antibody (blocking)
      1% BSA (stabilizer)
      0.1% cold fish skin gelatin (blocking)
      0.1% Triton X-100 (penetration enhancer)
      0.05% Tween 20 (detergent and surface tension reducer)
      0.05% sodium azide (preservative)
      Dissolve in 1X PBS
      Mix well and store at 40C.


      Avidin/Biotin Block:
      Avidin 0.001% in 1 X PBS
      Biotin 0.001% in 1 X PBS
      Store these blocking solution at 4oC.


      Primary Antibody Dilution Buffer:
      1%BSA (stabilizer and blocking)
      0.1% cold fish skin gelatin (blocking)
      0.05% sodium azide (preservative)
      0.01M PBS pH7.2

      Note: 1) Antibodies diluted using this buffer can be stored at 4°C for 6 months without reducing binding activity. 2) This buffer cannot be used for diluting HRP conjugated antibodies since sodium azide is an inhibitor of HRP.


      Peroxidase Blocking Solution (3% H2O2 in PBS):
      30% H2O2 2 ml
      1 X PBS - 18 ml
      Mix well and store at 4°C for up to 3 months.
      This solution is recommended for paraffin sections


      References:
      1. Shi SR, Chaiwun B, Young L, Cote RJ, Taylor CR. Antigen retrieval technique utilizing citrate buffer or urea solution for immunohistochemical demonstration of androgen receptor in formalin-fixed paraffin sections. J Histochem Cytochem. 1993 Nov; 41(11):1599-604. PubMed Abstract
      2. Kanai K, Nunoya T, Shibuya K, Nakamura T, Tajima M (1998) Variations in effectiveness of antigen retrieval pretreatments for diagnostic immunohistochemistry. Res Vet Sci. 64(1):57-61. PubMed Abstract
      3. Brown RW, Chirala R (1995) Utility of microwave-citrate antigen retrieval in diagnostic immunohistochemistry. Mod Pathol. 8(5):515-20. PubMed Abstract
      4. Morgan JM, Navabi H, Schmid KW, Jasani B. Possible role of tissue-bound calcium ions in citrate-mediated high-temperature antigen retrieval. J Pathol. 1994 Dec; 174(4):301-7. PubMed Abstract
      5. Pellicer EM, Sundblad A (1994) Antigen retrieval by microwave oven with buffer of citric acid. Medicina (B Aires). 54(2):129-32. PubMed Abstract
      6. Shi SR, Chaiwun B, Young L, Cote RJ, Taylor CR (1993) Antigen retrieval technique utilizing citrate buffer or urea solution for immunohistochemical demonstration of androgen receptor in formalin-fixed paraffin sections. J Histochem Cytochem. 41(11):1599-604. PubMed Abstract
      7. Brown, D., et al. (1996) Antigen retrieval in cryostat tissue sections and cultured cells by treatment with sodium dodecyl sulfate (SDS).Histochem Cell Biol 105:261–267.



      Protocols for ab92378 Anti-HNF4 antibody [EPR3648] and ab129189 Anti-NAPSIN A antibody [EPR6257]:


      1. Solutions and reagents

      1.1 Xylene
      1.2. Ethanol, anhydrous denatured, histological grade (100%, 95%, 70%, 50%)
      1.3. Washing buffer/TBST: 1X TBS/0.1% Tween-20, pH to 7.6.
      1.4. Distilled water (dH 2O)
      1.5. Antigen Retrieval Solution: 0.01M Sodium Citrate Buffer, pH 6.0
      To prepare Antigen Retrieval stock solutions:

      10X Stock: Dissolve 29.4 g sodium citrate trisodium salt dihydrate (C 6H 5Na 3O 7 2H 2O)
      in 1 liter of dH2O. Add 5mL Tween-20.

      1X Working Solution: Mix 200mL 10X stock with 1800mL dH2O; pH to 6.0

      1.6. 3% Hydrogen Peroxide
      1.7. Blocking Buffer: 10% serum in PBS (serum origin depends on the host of the secondary antibody)
      1.8. Primary Antibody Diluent: 5% serum in PBS (serum origin depends on the host of the secondary antibody)
      1.9. Hematoxylin
      1.10. Permanent Mounting Medium



      2. IHC Protocol

      2.1. Deparaffinization/Rehydration
      2.1.1. Heat slides in an oven at 65 °C for 1 hour.
      2.1.2. De-paraffinize/hydrate using the following series of washes: two Xylene washes (3 min each), followed by two 100% ethanol rinses (3 min each), followed by 95% ethanol, 70% ethanol, 50% ethanol, 30% ethanol, followed by TBST wash for 3 min on a shaker.


      2.2. Antigen Retrieval

      This is recommended Heat Induced Epitope Retrieval (HIER) using Decloaking Chamber/Pressure Cooker. Hot water bath or Microwave with temperature sensor can be also used (protocol would vary depending on the method used).

      2.2.1. Add 500 ml of dH 2O to Decloaker/Pressure Cooker.
      2.2.2. Immerse slides into staining dish containing Antigen Retrieval Solution. Place staining dish into decloaking chamber.
      2.2.3. Program to run for 30 seconds at 125° C, followed by 10 seconds at 90° C.
      2.2.4. Let it cool down to room temperature (10 - 20 minutes).
      2.2.5. Removes slides and rinse in TBST.
      2.2.6. Proceed to Staining step.


      2.3. Staining

      2.3.1. Wash slides with TBST for 3 min on a shaker.
      2.3.2. Inactivate endogenous peroxidase by covering tissue with 3% hydrogen peroxide for 5 min.
      2.3.3. Wash slides three times with TBST (3 min each on a shaker).
      2.3.4. Block slides with the blocking solution for 1 hour.
      2.3.5. Dilute primary antibody in primary antibody diluent per recommendation on data sheet.
      2.3.6. Apply primary antibody to each section and incubate overnight in the humidified
      chamber (4 °C).
      2.3.7. Wash slides three times with TBST (3 min each on a shaker).
      2.3.8. Apply to each section secondary HRP-conjugated anti-rabbit antibody diluted in the blocking solution per manufacturer's recommendation; incubate for 30 min at room temperature.
      2.3.9. Wash slides three times with TBST (5 min each on a shaker).
      2.3.10. Add freshly prepared DAB substrate to the sections and incubate until stain develops (generally 1 min).
      2.3.11. Rinse sections with water.
      2.3.12. Counterstain with Hematoxylin (generally 10 seconds).
      2.3.13. Rinse sections with water.
      2.3.14. Dehydrate samples using two washes with 100% Ethanol (3 min each), followed by two rinses with Xylene (3 min each).

      2.3.15. Mount coverslips on slides using permanent mounting medium.

      Read More

      Abcam Scientific Support

      Answered on Sep 27 2012

      Question

      I kindly ask you to inform me about the amount of reagent (Napsin A, Pax8 and HNF4) required for one analysis (or how many analysis can be done with 1 package of the mentioned reagents).

      Thank you in advance.

      Best regards,

      Read More

      Abcam community

      Verified customer

      Asked on Sep 25 2012

      Answer

      Thank you for your message which has been forwarded to the scientific support team.

      I am sorry it is difficult to let you know the amount of antibody required for one analysis. This will be very individual and will depend on:

      1. The dilution of antibody used, which will need to be optimized by the end user.

      2. The amount of antibody solution added to each sample.

      3. The application being used.

      I can suggest to use the following information from the online datasheets as a guide. This provides the amount of antibody provided in each vial and also the recommended dilutions, which will require optimization for individual experiments:

      ab129189 NAPSIN A 100 µl
      Recommended dilutions:
      WB: 1/1000 - 1/10000.
      IHC-P: 1/100 - 1/250.


      ab92378 HNF4 100 µl
      Reommended dilutions:
      WB: 1/1000 - 1/10000
      IP: 1/10 - 1/100.
      IHC-P: 1/100 - 1/250
      ICC: 1/100 - 1/250.
      Flow Cyt: 1/100.

      ab135618 PAX8 250 ul
      Recomended dilutions.
      WB: 1/100 - 1/500.
      IHC-P: 1/50 - 1/100.
      Flow Cyt: 1/10 - 1/50.

      I hope this will be helpful to you. If you require any further assistance, please let me know which application you are using, and how much antibody solution you will use for each application. If you have any further questions, please do not hesitate to let me know.

      Read More

      Abcam Scientific Support

      Answered on Sep 25 2012

      Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
      For licensing inquiries, please contact partnerships@abcam.com

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