Human Antibody Used On Human 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 screening human antibody on human tissues. GBI Labs Klear human kit is designed for staining human primary antibodies on human tissues without background staining. The Klear Human HRP-Polymer Detection kit provides special blocking buffers, polymeric HRP or AP-linked secondary antibody as well as human primer in a ready to use system. This technology requires an overnight pre-incubation with primary antibody that results in 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. Note: This kit is recommended for cytoplasmic and membrane bound antigens, other patterns of staining have not tested.

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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.

Day 1:

Dilute primary antibody in Human primer (Reagent 1) at user determined primary antibody concentration. Mix gently for 30 sec to 1min. Recommend only diluting amount needed for experiment. Place at 4oC overnight.

Day 2:

  1. Prepare slides as described above.

  2. 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).

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

    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. Heat Induced Epitope Retrieval (HIER) Pretreatment may be required for primary antibody. Detailed procedures refer to antibody supplier’s data.
  5. Wash the slide with PBS/0.05% tween20 (for HRP) or 1x TBS-T (For AP) for 2 min., 3 times.
  6. Remove the primary antibody mix from fridge and allow it to come to room temperature.
  7. Add Quenching Buffer (Reagent 2, 5x Concentration) into mixture. Incubate at room temperature for 15-30 min. Store at 4oC or on ice until you reach step 12.

    8. Note: Do not quench longer than 1hour. Recommend starting quenching after you add blocking buffer to your slides to get the timing correct.

    Blocking

  8. Add 2 drops or enough volume of Hu Blocking A (Reagent 3) to cover the tissue section completely and Incubate 30 min.
  9. Wash the slide with PBS/0.05% tween20 (for HRP) or 1x TBS-T (For AP) for 2 min., 3 times.
  10. Add 2 drops or enough volume of Hu Blocking B (Reagent 4) to cover the tissue section completely and Incubate 5 min.
  11. Wash the slide with PBS/0.05% tween20 (for HRP) or 1x TBS-T (For AP) for 2 min., 3 times.

    12. Primary Ab binding

  12. Add 2 drops or enough volume of mixture from step 7 {(Primary Ab) /(Reagent 1 Human Primer) /( Reagent 2 Quenching Buffer)} to cover the tissue section completely and Incubate 30-60 min. (Recommend 2 hours, but it will increase background)

    13. Note: Optimized incubation time should be tested. We find that incubating 2-4 hours at room temperature or overnight at 4oC works great without background

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

    14. 2nd Ab binding

  14. Apply 2 drops or enough volume of Human HRP Polymer (Reagent 5) to cover the tissue section completely and incubate 10 minutes.
  15. Wash the slide with PBS/0.05% tween20 (for HRP) or 1x TBS-T (For AP) for 2 min., 3 times.

    Detection with different substrates

    HRP AEC substrate

  1. Add 1 drop of AEC substrate (20X) into 1ml distilled water, mix well. Then add 1 drop of AEC Chromogen (20X) and 1 drop of Hydrogen Peroxide (20X) in the diluted substrate buffer. Mix well. Protect from light and use within 1 hour.
  2. Apply 2 drops (100μl) or enough of mixture to completely cover tissue. Incubate for about 5-10 min. Monitor the color development under microscope.
  3. Rinse with tap water for 1-2 min.

    4. Note: AEC is alcohol soluble, do not dehydrate! Use Aqueous Mount.

    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. Protect from light and use within 7 hours.
  2. Apply 2 drops (100μl) or enough of mixture to completely cover tissue. Incubate for about 5 min. Monitor the color development under microscope.
  3. Rinse with tap water for 1-2 min.

    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 (100μl) or enough volume of Fast Red solution to completely cover the tissue. Incubate for 10-20min, 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 μl Permanent Red Activator) into 1mL of Permanent Red substrate buffer and mix well. Add 10 μl Permanent Red Chromogen into the mixture and mix well.
  2. Apply 2 drops (100μl) 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)