This method is used to visualise the localisation and quantity of a protein of interest. The target protein is bound to by a specific primary antibody, which in turn is detected by a secondary antibody conjugated to a fluorophore. A fluorescent or confocal microscope is used to visualise the protein. Immunocytochemistry (ICC) differs from immunohistochemistry (IHC) in that the former is performed on samples of intact cells that have had most, if not all, of their surrounding extracellular matrix removed. In contrast, immunohistochemical samples are sections of biological tissue, where each cell is surrounded by tissue architecture and other cells normally found in the intact tissue. These differences cause the samples to be prepared differently. For ICC, the sample requires permeabilisation so that the antibodies can reach the intracellular targets. Depending on the thickness of the sample, IHC samples do not require this. Do you have a technical question? Get in touch: info@stjohnslabs.com Protocol series - http://www.stjohnslabs.com/services/video-protocol-series

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Immunohistochemistry Protocol
Tissue processing
Incubate slides sequentially into: 100% xylene, 50% Xylene, Anhydrous
ethanol for 15 min each, followed by anhydrous ethanol, 85% alcohol and
75% alcohol for 5 min each, then wash in distilled water.
Antigen retrieval
Incubate tissue slides with citric acid (pH6.0) antigen retrieval buffer and
microwave for antigen retrieval (heat until boiled and then stop heating) for
8min, heat slides on medium power for 7min. During this process keep the
slides from drying out, after a cooling down period at room temperature,
wash the slides with PBS on shaker for 5 min, repeat this process 3 times.
Inhibition of endogenous peroxidase
Place the slides in 3% Hydrogen peroxide solution, and incubate for 10 min
at room temperature without light exposure, once completed wash the
slides 3 times with PBS on a shaker for 5 mins.
BSA Blocking
Shortly after slides are dry, use a PAP pen to draw circles around the tissue
sections (and to prevent draining of the antibody solution), use BSA to cover
the tissue evenly, blocking for 30 min.
Primary antibody incubation
Remove the blocking solution, then add the 1:100-1:300 solution of primary
antibody/PBS on the slide, and incubated overnight at 4°C, add a small
amount of distilled water into the incubation box (to prevent evaporation of
antibody).

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Secondary antibody incubation
Wash the slides 3 times with PBS on a shaker for 5min, shortly after the slides
are dry, add the corresponding secondary antibody solution (HRP labelled),
covering the tissues, and incubate at room temperature for 30 min.
DAB staining
Wash the slides 3 times with PBS on a shaker for 5min, shortly after the slides
are dry add a fresh DAB staining buffer to the circles, the staining time
needs adjusting under a microscope (yellow-brown colour will represent a
positive result), stop the staining by washing the slides with water.
Haematoxylin staining
Use haematoxylin to counter-staining for 1min, and then wash the slides with
water, add 1% Hydrochloric acid and alcohol for several seconds and then
wash with water, use ammonia to reveal blue colour, and then flush with
water.
Desolation and Clearing
Incubate the slides sequentially into: 75% alcohol, 85% alcohol, Anhydrous
ethanol, Anhydrous ethanol and Xylene for 5 min each, shortly after slides
are dry, use neutral gum to seal the slides.
Visualization
Scan the slides and validate the results under a microscope.