Why are samples treated with hydrogen peroxide prior to staining with horseradish peroxidase?
Endogenous peroxidases will react with the substrate solution (hydrogen peroxide and chromogen, eg DAB), leading to false positives. This non-specific background can be significantly reduced by pre-treatment of the sample with hydrogen peroxide before incubation with HRP-conjugated antibody.
How do you prepare tissue samples for immunohistochemistry?
Frozen tissue can be prepared by immersing the tissue in liquid nitrogen or isopentane, or by burying it in dry ice. For frozen samples, a short fixation is done after freezing and sectioning.
What should I dilute primary antibody in IHC?
Apply primary antibody diluted in TBS with 1% BSA. Dilute the primary antibody to the manufacturer’s recommendations or to a previously optimized dilution. Most antibodies will be used in IHC-P at a concentration of 0.5–10 μg/mL.
Why is hydrogen peroxide used in immunohistochemistry?
Hydrogen peroxide (H2O2), a blocking agent in immunohistochemistry, is commonly used to block endogenous peroxidase acitivity. Pre-treatment with saturating amounts of hydrogen peroxide results in the irreversible inactivation of endogenous peroxidase.
How do you Deparaffinize a slide?
Deparaffinize in xylenes using three changes for 5 minutes each. Hydrate sections gradually through graded alcohols: wash in 100% ethanol twice for 10 minutes each, then 95% ethanol twice for 10 minutes each. Wash in deionized H2O for 1 minute with stirring. Aspirate excess liquid from slides.
What is immunocytochemical staining?
Listen to pronunciation. (IH-myoo-noh-SY-toh-KEH-mih-stree) A laboratory method that uses antibodies to check for certain antigens (markers) in a sample of cells. The antibodies are usually linked to an enzyme or a fluorescent dye.
What stains are used in immunohistochemistry?
Common counterstains include hematoxylin, eosin, nuclear fast red, methyl green, DAPI, and Hoechst fluorescent stain. The following representative example, Hoechst fluorescent dye was used as a counterstain for IHC detection of the protein, vimentin.
What are immunohistochemistry techniques?
Immunohistochemical techniques detect antigens in tissue sections by means of immunological and chemical reactions. This technique is highly sensitive and specific and can detect a wide variety of antigens in multiple animal species.
How do you dilute IHC antibodies?
Dilute the primary antibody in blocking solution (e.g. 3% BSA in PBS), then incubate for 1-2 hours at room temperature, or overnight at 39°F/4°C.
What is PBS in IHC?
IHC Washing Buffer – Phosphate Buffered Saline (PBS)