Mouse Antibody Used On Mouse Tissue


Antigen detection with primary antibody of the same species as the test tissue yields high background when indirect detection method is used. This severely limits the use of murine monoclonal antibody on mouse tissues. GBI Labs Klear mouse kit is designed for staining mouse antibodies on mouse tissues. The Klear Mouse HRP or AP-Polymer Detection kit uses a special blocking buffer, polymeric HRP or AP-linked secondary antibody as well as mouse antibody enhancer to detect mouse primary antibody that bound to the mouse tissue. This technology provides excellent sensitivity and high specificity. It is a biotin-free system, therefore, overcomes the non-specific staining caused by streptavidin/biotin system due to endogenous biotins. With Klear mouse system, the primary antibody will need to be screened at higher concentration (2-10 folds) than regular mouse detection kits.

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Protocol

Tissue Slide Preparation

  1. Fixation: To ensure the quality of the staining and obtain reproducible performance, user needs to supply appropriately fixed tissue and well prepared slides.
  2. Tissue needs to be adhered to the slide tightly to avoid tissue falling off.
  3. Paraffin embedded section must be deparaffinized with xylene and rehydrated with a graded series of ethanol before staining.
  4. Cell smear samples should be made into a monolayer as much as possible to obtain satisfactory results.
  5. Three control slides will aid the interpretation of the result: positive tissue control, reagent control (slide treated with Isotype control reagent), and negative control.
  6. Start staining procedures: DO NOT let specimen or tissue dry from this point on.

Staining

*HRP and AP require different blocking and wash reagents. Please refer to the correct label for the following steps.

    Blocking

  1. HRP: Apply 2 drops or enough volume of Peroxidase blocking reagent (3% H2O2 solution) to cover the tissue section and incubate for 10 mins.

    AP: Apply 2 drops or enough volume of phosphatase blocking reagent (Klear Dual Block- E36-xx) to cover the tissue section and incubate for 10 mins.

    Rinse the slide using distilled water and move to pretreatment step (optional).

  2. Heat Induced Epitope Retrieval (HIER) Pretreatment may be required for primary antibody. Detailed procedures refer to antibody supplier?s data.

  3. Wash the slide with PBS/0.05% tween20 (for HRP) or 1x TBS-T (For AP) 3 times for 2 minutes each.

    We recommend TBS-T to be used as the wash buffer for AP linked kit to get the highest sensitivity and clean background. Phosphate in the PBS-T may inhibitor the activity of the alkaline phosphatase. Note: 1X TBS-T =50mM Tris HCl, 150mM NaCl, 0.05% Tween-20 pH7.6.

  4. Add 2 drops or enough volume of MS blocking A (Reagent 1) to cover the tissue section completely and Incubate 30 minutes.
  5. Wash the slide with PBS/0.05% tween20 (for HRP) or 1x TBS-T (For AP) 3 times for 2 minutes each.
  6. Add 2 drops or enough volume of MS blocking B (Reagent 2) to cover the tissue section completely and Incubate 5 minutes.
  7. Wash the slide with PBS/0.05% tween20 (for HRP) or 1x TBS-T (For AP) 3 times for 2 minutes each.

    8. Primary Ab binding

  8. Apply 2 drops or enough volume of Primary antibody to cover the tissue section completely. Incubate in moist chamber for 30-60 minutes.
  9. Wash the slide with PBS/0.05% tween20 (for HRP) or 1x TBS-T (For AP) 3 times for 2 minutes each.

    10. 2nd Ab binding

  10. Apply 2 drops or enough volume of Polymer HRP or AP anti-mouse Antibody (Reagent 4) to cover the tissue section completely and incubate 15 minutes, longer incubation may increase background.
  11. Wash the slide with PBS/0.05% tween20 (for HRP) or 1x TBS-T (For AP) 3 times for 2 minutes each.

    Detection with different substrates

    HRP DAB substrate

  1. Add 1 drop or 2 drops (for higher sensitivity and contrast) of DAB Chromogen (20X) into 1ml of DAB substrate (20X). Mix well and store at 4oC. Protect from light and use within 4 hours.
  2. Apply 2 drops (100?l) or enough of mixture to completely cover tissue. Incubate for about 5 minutes. Monitor the color development under microscope.
  3. Wash with distilled water 3 times for 2 minutes each.

    AP Fast Red substrate

  1. Dissolve 1 tablet of Fast Red Tablet in 5ml Fast Red Substrate buffer, vortex until the tablet dissolved completely. Use within 1 hour.
  2. Apply 2 drops (100?l) or enough volume of Fast Red solution to completely cover the tissue. Incubate for 5-10min, observe appropriate color development.
  3. Rinse well with distilled water. (Fast Red is alcohol soluble; do not dehydrate.)

    AP Permanent Red substrate

  1. Add 200 ?l Permanent Red Activator into 1mL of Permanent Red substrate buffer and mix well. Add 10 ?l Permanent Red Chromogen into the mixture and mix well.
  2. Apply 2 drops (100?l) or enough volume of Permanent Red solution to completely cover the tissue. Incubate for 10 min, observe appropriate color development. To increase AP signal, aspirate or tap off chromogen and apply Permanent Red working solution again to completely cover the tissue for additional 5 to 10min.
  3. Rinse well with distill water.

    Counterstain

  1. Counterstain with 2 drops or enough volume to cover tissue completely and wait about 10-20 seconds.
  2. Wash thoroughly under tap water for 1-2 min.
  3. Put slides in PBS (for HRP) or TBS (for AP) until show blue color (about 30-60 seconds)
  4. Rinse well in distilled water

    Mounting

    Follow the manufacture data sheet procedure for mounting.

    Recommended product:

    • GB-Mount: Cat. No. E01-18 (18mL)
    • Simpo-Mount: Cat.No. E03-18 (18mL)