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What is the iso-IHC Kit?
The iso-IHC Kit is specifically designed for optimum immunohistochemical staining using mouse primary antibodies on mouse tissues and tissues from all other species. This system gives very low background, even on mouse tissues.
What is the principle of the iso-IHC Kit?
The iso-IHC Kit suppresses the background that results from the cross reactivity between the secondary anti-mouse antibody with endogenous immunoglobulins present in mouse tissues.
The key of the iso-IHC kit is that the primary antibody is labeled with an exclusive biotin labeling reagent, then mixed with a special blocking reagent that binds excess labeling reagent. The labeled antibody is then added to the tissue section and detected by the streptavidin-enzyme conjugate and substrate.
What is the protocol for the iso-IHC kit?
- Remove wax with EZ-DeWax™ Solution
- Retrieve antigen with Target Retrieval (optional)
- Inactivate endogenous peroxidase by Peroxide Block (for HRP)
- Minimize non-specific binding with Power Block™ Reagent
- Apply Labeled Antibody Mixture* and incubate
- Incubate with Streptavidin-Enzyme conjugate
- Develop color by adding Substrate Solution
- Counterstain with Hematoxylin or Nuclear Fast Red
- Mount the section with Mounting Medium
- Examine the slide under proper microscopy
* Labeled Antibody Mixture is prepared by mixing:
- Primary antibody
- Biotin Labeling Reagent
- MouseBlock Reagent.
What are the advantages iso-IHC Kit?
iso-IHC Kit is specifically designed for optimum immunohistochemical staining using mouse antibodies on mouse tissues. Also called the Mouse-on-Mouse kit, this self-contained kit includes all required pre-titrated reagents in precision dropper bottles. The kit can be used to stain other tissues as well.
- Very low background, even on mouse tissues
- Matched detection reagents and protocols for maximal signal-to-noise ratios
- Very high specificity inherent to the antibody-antigen and biotin-avidin interactions
- Very high sensitivity due to serial amplification
- Publication-quality results in approximately 2 hours
What IHC controls should I set up before I plan the experiment?
Negative Controls:
Labeled antibody/MouseBlock mixture is omitted.
Labeled antibody/MouseBlock mixture & avidin-enzyme conjugate are omitted.
Primary antibody is replaced with an isotype matched non-reactive antibody.
Positive Controls:
An antibody known to react with the test tissue replaces primary antibody.
Why does the negative control have staining?
- Insufficient blocking of endogenous biotin
- Bacterial contamination of water or reagents
- Excessive adhesive on slide
- Over-development of substrate
- Tissue is necrotic
Why do the slides have no staining?
- Reagents applied in wrong order or step omitted.
- Tissue may be overfixed or improperly fixed.
- Incorrect mounting medium.
- Tissue dried out during staining procedure.
- Incorrect primary antibody dilution
- Incorrect target retrieval procedure
Why do the slides have weak staining?
- Wash buffer not adequately drained after every wash step
- Inadequate incubation times
- Substrate/chromogen defective or contaminated
- Primary antibody overdiluted or may need longer incubation times
- Improper fixation or deparaffinization of tissue
- Low concentration of antigen
Why do the slides have excessive background?
- Inadequate rinsing of slides
- Reagents not properly diluted
- Over-development of substrate
- Incomplete deparaffinization
- Tissue dried out during staining procedure
- Inadequate blocking of endogenous biotin
- Inadequate blocking of endogenous peroxidase
- Diffusion of soluble antigen
- Primary antibody concentration too high
Why do the slides have spurious or erroneous staining?
- Primary antibody may contain cross-reacting antibodies
- Insufficient blocking of endogenous biotin or peroxidase
What references could you provide if I want to learn more about IHC?
Carson, F.L. (1990) Histotechnology: A Self-Instructional Text.
ASCP Press, Chicago.
Elias, J.M. (1990) Immunohistopathology: A Practical Approach to Diagnosis.
ASCP Press, Chicago.
Taylor, C.R. and Cote, R.J. (1994) Immunomicroscopy: A Diagnostic Tool for the Surgical Pathologist. W.B. Saunders Co., Philadelphia.
Shi, S.R., Gu, J., Kalra, K.L., Chen, T., Cote, R.J., and Taylor, C.R. (1995) Antigen retrieval technique: a novel approach to immunohistochemistry on routinely processed tissue sections.
Cell Vision 2: 6-22.
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