Immunohistochemistry (IHC) Protocol — Paraffin Sections

 

Experimental Protocol

Specimen Fixation

Cut fresh tissue into small pieces (approximately 1.5 cm × 1.5 cm × 0.3 cm), fix in 4% paraformaldehyde (or formalin) overnight. Generally, fixation time should not exceed 48 hours.

Dehydration, Paraffin Embedding and Sectioning

Dehydration:

To minimize rapid tissue shrinkage, proceed in ascending concentrations from low to high: 70%, 85%, 95%, to absolute ethanol (anhydrous ethanol), 1 hour for each step, ensuring complete dehydration. Fixed tissue specimens must be cleared of residual fixative and crystalline precipitates, otherwise subsequent staining results may be compromised.

Clearing:

The process of immersing specimens in clearing agent is termed "clearing." Xylene is commonly used as the clearing agent, which serves as a solvent for paraffin. Absolute ethanol is immiscible with paraffin; therefore, a solvent miscible with both ethanol and paraffin is required to replace ethanol in the tissue. Transfer dehydrated tissue blocks into xylene for approximately 20 minutes until the tissue appears translucent. Avoid over-clearing, as this will cause tissue brittleness and fragility.

Infiltration:

First immerse tissue blocks in a 1:1 mixture of molten paraffin and xylene for 1 hour, then transfer successively to two changes of molten paraffin for approximately 1 hour each.

Embedding:

Apply a small amount of hot paraffin wax to rapidly attach the bottom to the embedding cassette, then place on a cooling platform to allow the paraffin to solidify.

Sectioning:

The sharpness of the microtome knife and appropriate paraffin block hardness directly affect section quality. Paraffin block hardness can be modified by placing it in cold water. Typically, section thickness is 3-5 μm. Gently transfer sections with a brush and float them in a 37°C water bath for expansion.

Mounting:

A constant temperature water bath is generally used, controlled at approximately 37-40°C, which facilitates the expansion of paraffin sections.

Baking:

After tissue sections are fully expanded, retrieve slides and place in a 60°C oven for baking (baking time varies according to tissue type; generally 30 minutes for most tissues, but bone tissue requires extended baking up to 1 hour). Deparaffinization can then proceed.

Deparaffinization and Rehydration

Xylene (5 min) — Xylene (5 min) — Xylene (5 min) — 100% Ethanol (5 min) — 100% Ethanol (5 min) — 95% Ethanol (3 min) — 85% Ethanol (3 min) — 75% Ethanol (3 min) — Deionized Water (5 min).

Antigen Retrieval

Place deparaffinized sections into antigen retrieval solution (select sodium citrate or EDTA according to primary antibody datasheet requirements). Perform heat-induced epitope retrieval (HIER) in a pressure cooker: heat until full pressure is reached, then continue heating for 5 minutes. Turn off heat, wait 10 minutes before removing the staining container, and cool at room temperature for 30 minutes. Alternatively, perform microwave antigen retrieval: place deparaffinized and rehydrated slides in a retrieval chamber, immerse in 1× working solution of antigen retrieval buffer, and boil at high power in a microwave. Maintain at low power for 15 minutes (note: replenish liquid to prevent excessive evaporation causing dry sections). Remove and allow to cool naturally to room temperature.

Cell Permeabilization (Required for Cytoplasmic and Nuclear Targets)

For paraffin sections approximately 4 μm thick, permeabilization may be omitted as cells have already been sectioned. For thick sections >10 μm, use 0.1% Triton X-100 for permeabilization for 5-10 minutes. The purpose is to enable antibodies to fully enter cells for binding reactions.

Inactivation of Endogenous Peroxidase and Biotin

Draw a hydrophobic barrier around tissue sections, wash slides 3 times in PBST for 3 minutes each. Add 100 μL 3% H2O2 (volume should cover the sample area, adjustable as needed) per section, incubate at room temperature for 10 minutes. Wash with PBST for 3 minutes × 3 times. The purpose is to eliminate endogenous peroxidase activity.

Serum Blocking

Add 100 μL 5% normal goat serum blocking solution (volume should cover the sample area, adjustable as needed) per slide, incubate at room temperature for 30 minutes, then remove blocking solution. The purpose is to bind non-specific proteins, providing blocking action to reduce non-specific staining.

Antibody Incubation

Incubate with 100 μL primary antibody at room temperature in a humidified chamber for 1 hour or overnight at 4°C. Wash with PBST for 3 minutes, 3 times. Discard PBST and add 100 μL secondary antibody, incubate for 30 minutes to 1 hour. Wash with PBST for 3 minutes, 3 times.

DAB Chromogenic Detection

Add 100 μL DAB chromogenic substrate solution, develop for 5 minutes, terminate the chromogenic reaction with tap water. Both background intensity and specific staining intensity can be controlled by DAB incubation conditions. DAB development time is not fixed and is primarily controlled under the microscope; wash when specific staining is strong and background staining is minimal.

Counterstaining

Counterstain with Harris hematoxylin for 3 minutes (1 minute for tumor tissues only), rinse with running tap water with agitation, differentiate in 1% acid alcohol for 1 second, briefly wash with tap water, blue in running tap water for 5 minutes (or blue with 2% ammonia water for 1-2 seconds). The purpose is to create cellular outlines for better localization of target proteins; Harris hematoxylin (nuclear stain) is commonly used.

Dehydration, Clearing and Mounting

For long-term preservation, neutral mounting medium is generally used for coverslipping, avoiding bubble formation. The method is to directly apply one drop of mounting medium onto the tissue on the slide, then cover with a coverslip. After mounting, bake at 60°C for 20 minutes.