What is Immuno Histo Chemistry? How to search IHC Laboratory for research samples ?

 

Immuno Histo Chemistry (IHC) 

IHC is a kind of staining process to detect carcinomas performed on paraffin-embedded tissue. In oncology, immunohistochemistry is now widely used to distinguish sarcomas from carcinomas; malignant lymphomas from epithelial neoplasms; and malignant melanomas from both carcinomas and sarcomas.

Why the IHC test is performed?

IHC is a special application of monoclonal as well as polyclonal antibodies to the diagnosis of cancers; specific tumor antigens are expressed de novo or up-regulated in certain types of cancers.

What are the IHC Markers?

IHC markers are monoclonal antibodies used to stain specific proteins in tissue sections. The antibody binds to the protein gives a color reagent stains the protein, in fact; the protein is present in the tissue. The proteins that we search to indicate the differentiation of the tumor and co-operate us to render a diagnosis. Identification of specific proteins is often required for tumors that might not be readily classified on routine sections alone (e.g. spindle cell tumors, round cell tumors).

We usually need to test for the presence of antibodies to the following proteins:

·          SMA or smooth muscle actin 

·         Desmin 

·         S-100 

·         HMB-45 

·         Cytokeratins 

·         Vimentin 

·         LCA or leukocyte common antigen 

·         CD20 

·         CD3 

·         CD34

What are the IHC procedures?

The basic steps of the IHC-P protocol are as follows:

1.  Fixing and embedding the tissue

 2. Cutting and mounting the section

3. Deparaffinizing and rehydrating the section

4. Antigen retrieval

5. IHC staining

6. Counterstaining (if desired)

7. Dehydrating and stabilizing with mounting medium

 8. Viewing the staining under the microscope

 

Note: Deparaffinization Before proceeding with the staining protocol, the slides must be deparaffinized and rehydrated. Incomplete removal of paraffin can cause poor staining of the section.

Reagents and types of equipment:--

1. Formalin-fixed, paraffin-embedded tissue sections/PLL coated tissue slides
2. 0.01 M phosphate-buffered saline, pH 7.4 (PBS) 
3. Bovine serum albumin (BSA)
4. Diluent: 1% BSA in PBS
5. Xylene
6. Ethanol 0.1% Trypsin in PBS or Trypsin tablet in 4mM CaCl₂, 200 mm Tris, pH 7.7, or 0.1% Protease in PBS.
7. Microwave antigen retrieval solution: 10 mM sodium citrate buffer pH 6.0, 1 mM EDTA pH 8.0
8. Primary antibody
9. Option 1: Biotinylated secondary antibody and ExtrAvidin-Peroxidase or ExtrAvidin-Alkaline Phosphatase, or Option 2: Secondary antibody conjugated to peroxidase or alkaline phosphatase
10. When using peroxidase-conjugated secondary antibodies or ExtrAvidin-Peroxidase: 3% hydrogen peroxide (in deionized water, freshly prepared from 30% stock).
11. Enzyme substrate: depending on the enzyme and color needed.
12. For peroxidase: AEC Staining Kit, DAB
13. Mayer's hematoxylin staining
14. Microscope

IHC Protocol Steps

1.   Slide Preparation

16.   Primary Antibody Reaction

17.   Secondary Antibody Reaction

18.   Subtract Preparation

19.   Development

20.   Counterstaining

 

Deparaffinization and Rehydration

Deparaffinization before proceeding with the staining protocol, the slides must be deparaffinized and rehydrated. Incomplete removal of paraffin can cause poor staining of the section.

1. Place the slides in a 56-60 °C oven for 15 min. (Caution: Oven temperature must not exceed 60 °C).
2. Transfer to a xylene bath and perform two changes of xylene for 5 min. each.
3. Shake off excess liquid and rehydrate slides in two changes of fresh absolute ethanol for 3 min. each.
4. Shake off excess liquid and place slide in fresh 90% ethanol for 3 min.
5. Shake off excess liquid and place slide in fresh 80% ethanol for 3 min.
6. Rinse the slides in gently running tap water for 30 seconds (avoid a direct jet which may wash off or loosen the section).
7. Place in PBS wash bath for further rehydration (30 min. at room temperature)

Research Projects for IHC

We have a laboratory facility to perform the above-mentioned IHC procedures for any tissue sample. Please give your response in the comment box. 

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