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Paraffin-Embedded Tissue Immunohistochemistry

Introduction
Immunohistochemistry (IHC) is a commonly used technique to monitor protein expression and localization in the context of tissue morphology. It uses antibodies to detect and analyze protein expression while maintaining the composition, cellular characteristics, and structure of native tissue. Chemical fixation locks molecular interactions within and between cells into place. Tissue samples may be embedded in paraffin wax or frozen before being cut into thin slices and mounted onto slides for analysis. Tissue collection, preservation, and fixation can vary greatly depending on the sample or the target of interest. IHC identifies the presence and pattern of a protein's expression in a biological sample through specific antibody binding. The precise binding that occurs between an antibody and its epitope allows detection of highly specific amino acid sequences within a protein, targeting defined regions, domains, or cleavage products. Antibodies can also detect specific post-translational modifications (PTM) on a protein.

Applications
IHC is used for disease diagnosis, biological research, and in drug development. For example, using specific tumor markers, physicians use IHC to diagnose if a tumor is benign or malignant, to determine its stage and grade, and to identify the cell type and origin of a metastasis in order to find the site of the primary tumor. A variety of other non-neoplastic diseases and conditions are diagnosed using IHC as a primary tool or as a confirmatory procedure. In a research context, IHC can be used alone or in conjunction with other analytical techniques to study, for example, normal tissue and organ development, pathological processes, wound healing, cell death and repair, and many other fields. IHC is also used in drug development to test drug efficacy by detecting either the activity or the up- or down-regulation of disease markers in the target tissues and elsewhere.

Sample Preparation
1
Place paraffin infiltrated tissue in a mold with a small volume of liquid paraffin. Cool briefly to immobilize the tissue. Place the base of a cassette on top of the mold. Fill with liquid paraffin, and then cool.


2
Cut thin slices on a microtome, and float sections in a water bath.


3
Mount sections on to charged slides and dry overnight. (Charged slides help the section to stick to the plate.)


Deparaffinization/Rehydration
NOTE: To perform antibody staining, paraffin wax must be removed from the sample and the sample must be rehydrated.Excessive drying can lead to inconsistent staining.

1
To remove paraffin wax, place sections in fresh xylene containers for 3 minutes each.


2
To begin rehydration process, place sections in two containers of 100% ethanol for 3 minutes each.


3
Incubate sections in two containers of 95% ethanol for 1 minute each.


4
Place sections in 70% ethanol for 1 minute.


5
To complete the rehydration process, wash sections twice in dH2O for 5 minutes each.


Antigen Retrieval
NOTE: Refer product datasheet for antibody-specific recommendation for the unmasking solution. Over or under heat can cause inconsistent staining.

Citrate Buffer
1
Heat slides in 1X citrate solution until boiling is initiated; follow with 20 minutes at sub-boiling temperature (95℃-98℃). Cool slides on bench top for 20 minutes.


EDTA
1
Bring slides to a boil in 1mM EDTA, pH 8.0; follow with 15 minutes at a sub-boiling temperature. (No cooling is necessary.)


TE
1
Bring slides to a boil in 10mM Tris/1mM EDTA, pH 9.0; maintain the sub-boiling temperature for 18 minutes. Cool at room temperature for 30 minutes.


Staining
NOTE: Use volumes necessary to cover the entire section.

1
Wash sections in dH2O three times for 5 minutes each.


2
Incubate sections in 3% hydrogen peroxide for 10 minutes. (Note: This step is important to quench endogenous peroxidase activity, which will help reduce high background staining)


3
Wash sections in dH2O two times for 5 minutes each.


4
To prevent non-specific binding, block each section with preferred blocking solution for 30 minutes at room temperature. (Use serum from the same species as the source of the secondary antibody.)


5
Remove blocking solution and add primary antibody at recommended dilution, in suggested diluent to each section.


6
Remove blocking solution and add primary antibody at recommended dilution, in suggested diluent to each section.


7
Incubate overnight at 4°C. (Incubation period should be followed as per the recommendation.)


8
Remove antibody solution and wash sections with wash buffer three times for 5 minutes each.


9
Cover section with secondary antibody conjugated with HRP for 60 minutes at room temperature.


10
Wash sections three times with wash buffer for 5 minutes each.


11
Cover section with the ready-to-use Stayright™ Purple solution. Incubate at room temperature for 5 - 15 minutes. (Optimal development times should be determined for each application.)


12
Immerse slides in dH2O to stop the development. Wash with dH2O for 5-10 minutes.


13
If desired, counterstain sections.


14
After counterstain, wash sections in dH2O two times for 5 minutes each.


Dehydration and Mounting

1
Place sections in 70% ethanol for 2 minutes.


2
Place sections in two containers of 95% ethanol for 1 minute each.


3
Place sections in two containers of 100% ethanol for 1 minute each.


4
Place sections in two washes of xylene for 1 minute each.


5
Mount sections with coverslips and organic permanent aqueous mounting medium, being careful to avoid leading any air bubbles.


6
Allow some time to mounting medium to set and view slides on microscope.