TITLE: Immunohistochemistry Protocol for Frozen Sections
AUTHORS: Kelsey Knight

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The following is a general procedure guide for preparation and staining of acetone-fixed frozen tissues using a purified, unconjugated primary antibody, biotinylated secondary antibody and streptavidin-horseradish peroxidase (SAv-HRP) and DAB detection system. Because each antigen differs in terms of requirement for fixation, amplification step, etc., it is not possible to write an inclusive protocol that will work for all antigens. The user must determine optimal conditions for each antigen of interest. Many protocols for staining individual antigens, as well as useful tips and troubleshooting guides for immunohistochemistry, can be found at the IHC World web site (</span><a href="https://www.ihcworld.com" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">https://www.ihcworld.com/</span></a><span>).</span></div></div>

[STEPS]
?. [Prepare frozen tissue sections:]
Place a freshly dissected tissue block (
?. [Prepare frozen tissue sections:]
Cover the entire tissue block with cryo-embedding media (e.g. OCT).
?. [Prepare frozen tissue sections:]
Slowly place the base mold containing the tissue block into liquid nitrogen till the entire tissue block is submerged into liquid nitrogen to ensure tissue is frozen completely.
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Store the frozen tissue block at -80°C until ready for sectioning.
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Transfer the frozen tissue block to a cryotome cryostat (e.g. -20°C) prior to sectioning and allow the temperature of the frozen tissue block to equilibrate to the temperature of the cryotome cryostat.
?. [Prepare frozen tissue sections:]
Section the frozen tissue block into a desired thickness (typically 5-10 µm) using the cryotome.
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Place the tissue sections onto glass slides suitable for immunohistochemistry (e.g. Superfrost).
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Dry the tissue sections overnight at room temperature. Sections can be stored in a sealed slide box at -80°C for later use.
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Fix the tissue sections with a suitable fixative. One of the commonly used fixation methods for frozen tissue sections is to immerse the slides in pre-cooled acetone (-20°C) for 10 min.
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Pour off the fixative and allow acetone to evaporate from the tissue sections for
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Rinse the slides in 300 ml of 10mM phosphate buffered saline (PBS) at a neutral pH for 2 changes, 5 min each.
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Incubate the slides in 0.3% H2O2solution in PBS at room temperature for 10 min to block endogenous peroxidase activity.
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Rinse the slides in 300 ml PBS for 2 changes, 5 min each.
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Optional: Add 100 µl blocking buffer (e.g. 10% fetal bovine serum in PBS) onto the sections of the slides and incubate in a humidified chamber at room temperature for 1 hour.
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Drain off the blocking buffer from the slides.
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Apply 100 µl an appropriately diluted primary antibody (in antibody dilution buffer, e.g. 0.5% bovine serum albumin in PBS) to the sections on the slides and incubate in a humidified chamber for 1 hour at room temperature or overnight at 4°C.
?. [Immunostain frozen tissue sections:]
Rinse the slides in 300 ml PBS for 2 changes, 5 min each.
?. [Immunostain frozen tissue sections:]
Apply 100 µl an appropriately diluted biotinylated secondary antibody (using the antibody dilution buffer) to the sections on the slides and incubate in a humidified chamber at room temperature for 30 min.
?. [Immunostain frozen tissue sections:]
Rinse the slides in 300 ml PBS for 2 changes, 5 min each.
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Add 100 µl pre-diluted Sav-HRP conjugates (using the antibody dilution buffer) to the sections on the slides and incubate in a humidified chamber at room temperature for 30 min (keep protected from light).
?. [Immunostain frozen tissue sections:]
Rinse the slides in 300 ml PBS for 2 changes, 5 min each.
?. [Immunostain frozen tissue sections:]
Apply 100 µl DAB substrate solution (freshly made just before use: 0.05% DAB - 0.015% H2O2in PBS) to the sections on the slides to reveal the color of the antibody staining. Allow the color development for Caution: DAB is a suspected carcinogen. Handle with care. Wear gloves, lab coat and eye protection.
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Wash slides in 300 ml PBS for 2 changes 5 min each.
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Optional: Counterstain slides by immersing sides in Hematoxylin (e.g. Gill's Hematoxylin) for 1-2 min.
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Rinse the slides in running tap water for > 15 min.
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Dehydrate the tissue slides through 4 changes of alcohol (95%, 95%, 100% and 100%), 5 min each.
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Clear the tissue slides in 3 changes of xylene and coverslip using mounting solution (e.g. Permount). The mounted slides can be stored at room temperature permanently.
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Observe the color of the antibody staining in the tissue sections under microscopy.