Immunohistochemistry Protocol

(For Formalin-fixed Paraffin Embedded Tissue Samples)

Immunohistochemistry (IHC) is the process of detecting antigens (e.g. proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. IHC can be used to detect protein expression in formalin-fixed paraffin-embedded (FFPE) tissue samples and widely used in pathology, particularly in the subspecialties of oncologic pathology, neuropathology, and hematopathology. This protocol is just for reference only. At Creative Bioarray, we can provide services for entire IHC process with unprcedented analytical accuracy, you can find more at Creative Bioarray’s Histology Services.

ICH-Assay Work Flow

Deparaffinization Rehydration

1)	Sequentially immerse paraffin sections into: a. 90% dimethybenzene (for 5 min); b. 95% dimethybenzene (for 5 min); c. 100% dimethybenzene (for 5 min); d. 90% ethanol (for 5 min); e. 95% ethanol (for 5 min); f. 100% ethanol (for 5min).
2)	Rinse slides twice in water for 5 min to remove ethanol.

Antigen Retrieval (Microwave Method)

1)	Immerse slides in 3% H₂O₂ for 5 minutes.
2)	Wash slides twice (2x) with distilled water (for 3-5 min).
3)	Immerse slides in the working citrate buffer and cover with a lid.
4)	Heat the buffer in microwave and turn it off when the buffer has boiled.
5)	Remove from heat and let it stand at room temerature for 20 minutes.
6)	Wash the slides 1X to 2X with PBS.
7)	Remove the liquid and use a pen to circle around the tissue.

Blocking

1)	Add 5% BSA blocking solution to the HIER treated samples.
2)	Incubate the samples at 37°C for 30 min.

Primary Antibody Icubation

1)	Dilute the primary antibody to the recommended concentration by the antibody manufactuer.
2)	Add the diluted antibody to the samples and incubate overnight at 4°C or 37°C for 1h.
3)	Wash slides three times (20 min) in 2X PBS each on the shaker.

Secondary Antibody Incubation and Detection

1)	Dilute secondary antibody with antibody diluent to the recommended concentration by the antibody manufactuer.
2)	Add the dilute antibody to the samples and incubate at 37°C for 30 min.
3)	Wash slides three times (20 min each) in 2X PBS on the shaker.
4)	Add Strept-Avidin Bion Complex (SABC) HRP- or AP-conjugated reagents to the samples. Incubate the samples at 37°C for 30 min.
5)	Wash the slides three times (20 min each) in 3X PBS each on the shaker.
6)	Add an enough amount of DAB reagent to the sampes and incubate in dark at room temperature for 10 to 30 min.
7)	Wash the samples with distilled water.
8)	Check the tissue staining intensity under a bright-field microscope.

For research use only. Not for any other purpose.