Cytokeratin 19

A. Dewaxing and rehydration

1. Place tissue sections in two baths of fresh xylene, 10 minutes each.
2. Place tissue sections in absolute ethanol, 95% ethanol, and 85% ethanol, 5 minutes each.
3. Gently rinse with distilled water for 1 minute, then keep the slides in distilled water until retrieval.

B. Antigen retrieval (pressure cooker)

1. Add prepared Tris-EDTA antigen retrieval solution (pH 9.0) to a pressure cooker. Place dewaxed, rehydrated sections on a heat-resistant plastic rack and immerse them completely in the retrieval solution.
2. Close the lid, lock the pressure valve, and heat on high (1600 W / 210°C) until steaming. Time 2 minutes from the start of steaming and reduce to medium heat (800 W / 130°C). When timing ends, turn off the hot plate, move the cooker off the heat, and cool naturally for 5 minutes.
3. Move the cooker to a sink, run water over the lid until the lock pin drops, turn off the water, remove the pressure valve, and open the lid carefully (watch for steam). Slowly add running water into the cooker (do not aim the stream at the sections).
4. When the temperature is below 40°C (water no longer feels hot), remove the rack and immediately immerse it in distilled water (avoid drying). Gently rinse with distilled water twice for 3 minutes each, then keep the slides in distilled water until blocking.

(Note: After retrieval, sections dry easily. In later steps, whenever you shake off excess liquid, do not let the tissue dry, or staining will be affected.)

C. Block endogenous peroxidase

1. Shake off excess liquid and immediately immerse slides in peroxidase blocker (3% H2O2) for 10 minutes.
2. After blocking, gently rinse with distilled water twice for 3 minutes each, then keep the slides in distilled water until circling.

D. Pap-pen circling

Shake off or carefully blot excess liquid around the tissue. Circle the tissue with an IHC pap pen, 2–3 mm from the tissue edge, enclosing the entire specimen. Gently rinse with distilled water twice for 3 minutes to remove excess oil, then keep the slides in PBS until primary antibody incubation.

E. Apply primary antibody or control reagent

1. Shake off excess liquid. When the pap-pen circle is fully visible, apply primary working solution to cover the tissue completely. Incubate 30 minutes at room temperature in a humid chamber.
2. After incubation, rinse with PBS 2–3 times, then immerse in a PBS bath 3 times for 2 minutes each.

(Note: When rinsing with PBS, do not aim the stream directly at the tissue surface, or sections may detach.)

F. Apply HRP-conjugated secondary polymer

1. Shake off excess liquid. When the pap-pen circle is fully visible, apply one-step secondary reagent to cover the tissue completely. Incubate 20 minutes at room temperature in a humid chamber.
2. After incubation, rinse with PBS 2–3 times, then immerse in a PBS bath 3 times for 2 minutes each.

G. Apply DAB chromogen

1. Shake off excess liquid. Apply freshly prepared DAB to cover the tissue completely. Incubate 5 minutes at room temperature in a humid chamber.
2. After DAB incubation, tap residual DAB onto a tissue, then rinse thoroughly in distilled water twice for 3 minutes to stop staining.

H. Counterstain and bluing

1. After thorough rinsing with distilled water, counterstain in hematoxylin for 8–10 seconds, then rinse with distilled water.
2. Blue in 1% lithium carbonate for 8–15 seconds, then rinse with distilled water.

(Note: Adjust counterstain time to the strength of the hematoxylin so nuclei appear light to dark blue. Over- or under-staining can affect interpretation.)

I. Dehydration, clearing, and mounting

1. Shake off excess liquid. Place sections in 85% ethanol, 95% ethanol, and absolute ethanol, 3 minutes each.
2. After dehydration, place sections in fresh xylene for 5–10 minutes.
3. When sections appear translucent, mount with neutral balsam and a coverslip.

J. Interpretation

IHC results must be observed and interpreted under a light microscope by experienced professionals.

A. False negative

A false negative means the target antigen should be present, but the sample shows no staining after testing.

1. Sample and antigen

1. Delayed fixation after excision lets endogenous enzymes degrade the target antigen. (Place large specimens in fixative within 30–60 min and needle biopsies within 1–2 min; adjust to the specimen.)
2. Over-fixation fully masks epitopes by excessive cross-linking. (Fix large specimens 12–18 h and needle biopsies 4–6 h; adjust to the specimen.)
3. Sections that are too thin contain little target antigen. (Cut paraffin sections at 3–5 μm; 3 μm is recommended.)

2. Reagent preparation

1. Reagents lose activity from expiry, incorrect storage temperature, or freeze–thaw cycles. (Check expiry before use; store at 2–8°C after use; aliquot to avoid freeze–thaw. Prepare retrieval and DAB solutions fresh.)
2. The wrong primary antibody is used, so the detected antigen does not match the target. (Verify name, lot, clone, and dilution before use.)
3. Over-dilution of the primary leaves too little antibody for the secondary to bind. (Use the datasheet dilution; verify other ratios with a titration.)
4. Secondary species reactivity does not match the primary host, so the two cannot bind. (Choose the secondary by the primary host species.)

3. Procedure

1. Incorrect retrieval conditions, too little time, insufficient temperature, or low pH leave epitopes closed. (Follow the datasheet retrieval method; verify alternatives experimentally.)
2. Sections dry after retrieval and epitopes re-close. (Keep sections moist after retrieval; avoid drying.)
3. Primary incubation, secondary incubation, or DAB development is too short. (Follow the datasheet times.)
4. The order of retrieval → primary → secondary → DAB is wrong or a step is skipped. (Follow the datasheet sequence.)
5. Too many or overly forceful distilled-water or PBS washes strip bound antibody. (Follow the datasheet wash count and time; do not jet the stream onto the section.)

B. False positive

A false positive means the target antigen should be absent, but the sample stains after testing.

1. Sample and antigen

1. Under-fixation lets the target antigen diffuse into surrounding tissue. (Fix large specimens 12–18 h and needle biopsies 4–6 h; adjust to the specimen.)
2. Necrotic areas break down and release non-target peptide fragments that bind antibody non-specifically. (Avoid specimens with large necrotic areas.)

2. Reagent preparation

1. Primary or secondary antibodies denature and aggregate after freeze–thaw or long room-temperature storage, forming protein clumps that adsorb non-specifically. (Store at 2–8°C; aliquot to avoid freeze–thaw.)
2. Primary concentration is too high, so excess antibody binds non-target sites. (Use the datasheet dilution; verify other ratios with a titration.)

3. Procedure

1. Retrieval that is too long, too high in pH, or too concentrated over-exposes epitopes and can damage tissue, creating non-target sites. (Follow the datasheet retrieval method; verify alternatives experimentally.)
2. Blocking is omitted or incomplete, so endogenous peroxidase reacts directly with DAB. (Immerse sections in 3% H2O2 for 10 min to block endogenous peroxidase.)
3. Incubation that is too long or too hot concentrates reagents and causes non-specific adsorption. (Incubate at room temperature for the datasheet times.)
4. The pipette tip is not changed between different primaries, or the wrong primary is added to the same slide, causing cross-contamination. (Change tips after each transfer. Keep different conditions in separate work areas.)
5. Too few or too gentle distilled-water or PBS washes leave free antibody behind. (Follow the datasheet wash count and time; wet all sections thoroughly.)

4. Interpretation

1. Endogenous pigments such as lipofuscin or hemosiderin are mistaken for positive signal. (Run a negative control on a serial section of the same specimen.)
2. Residual normal positive cells in tumor tissue are mistaken for tumor positivity. (Distinguish by cell morphology.)
3. Macrophages in normal tissue phagocytose target protein from positive cells and stain. (Distinguish by cell morphology.)
4. Tumor cells that invade normal tissue phagocytose target protein and stain. (Staining is uneven, often irregular vacuolar or granular “inclusion-like” deposits.)

C. Weak staining

1. Sample and antigen

1. The specimen itself has very little target antigen. (Use another specimen, or increase retrieval and incubation intensity.)
2. Prolonged storage has reduced target antigen. (Use another specimen, or increase retrieval and incubation intensity.)
3. Delayed or excessive fixation after excision damages target epitopes. (Place large specimens in fixative within 30–60 min and needle biopsies within 1–2 min; fix large specimens 12–18 h and needle biopsies 4–6 h; adjust to the specimen.)
4. Tumor or lesional tissue is heterogeneous, with positivity only in local areas. (Sample different regions of the tumor separately.)

2. Reagent preparation

1. Reagents lose activity from expiry, incorrect storage temperature, or freeze–thaw cycles. (Check expiry before use; store at 2–8°C after use; aliquot to avoid freeze–thaw. Prepare retrieval and DAB solutions fresh.)
2. Over-dilution of the primary leaves too little antibody for the secondary to bind. (Use the datasheet dilution; verify other ratios with a titration.)

3. Procedure

1. Retrieval that is too short, too cool, or too low in pH fails to open epitopes fully. (Follow the datasheet retrieval method; verify alternatives experimentally.)
2. Sections dry after retrieval and epitopes re-close. (Keep sections moist after retrieval; avoid drying.)
3. Primary incubation, secondary incubation, or DAB development is too short or too cold. (Incubate at room temperature for the datasheet times.)
4. Slides are not level during incubation, so antibody runs off. (Keep slides level during incubation.)
5. Too many or overly forceful distilled-water or PBS washes strip bound antibody. (Follow the datasheet wash count and time; do not jet the stream onto the section.)
6. Too much residual wash liquid further dilutes the next reagent. (Shake off or blot excess liquid before applying reagent, but do not let the tissue dry.)

D. Overstaining

1. Sample and antigen

1. The specimen itself has very high target antigen. (Use a higher primary dilution after verifying with a titration.)
2. Tumor or lesional tissue is heterogeneous, with locally high antigen density. (Sample different regions of the tumor separately.)
3. Under-fixation leaves abundant free target antigen exposed. (Fix large specimens 12–18 h and needle biopsies 4–6 h; adjust to the specimen.)
4. Thick sections greatly increase target antigen per unit area. (Cut paraffin sections at 3–5 μm; 3 μm is recommended.)

2. Reagent preparation

1. Excess primary concentration saturates specific signal. (Use the datasheet dilution; verify other ratios with a titration.)
2. Deteriorated DAB has abnormally high catalytic activity. (Prepare DAB fresh and check expiry before use.)

3. Procedure

1. Retrieval that is too long, too high in pH, or too concentrated over-exposes epitopes. (Follow the datasheet retrieval method; verify alternatives experimentally.)
2. Blocking is omitted or incomplete, so endogenous peroxidase reacts directly with DAB. (Immerse sections in 3% H2O2 for 10 min to block endogenous peroxidase.)
3. Incubation that is too long or too hot drives specific binding past saturation. (Incubate at room temperature for the datasheet times.)
4. Too few or too gentle distilled-water or PBS washes leave free antibody behind. (Follow the datasheet wash count and time; wet all sections thoroughly.)

E. Patchy staining

Patchy staining is an abnormal pattern in which positive signal appears as scattered spots rather than a uniform distribution.

1. Sample and antigen

1. Under-fixation lets antigen diffuse; over-fixation destroys epitopes; both can yield patchy staining. (Fix large specimens 12–18 h and needle biopsies 4–6 h; adjust to the specimen.)
2. Necrotic areas break down and release non-target peptide fragments that bind antibody non-specifically. (Avoid specimens with large necrotic areas.)
3. Uneven section thickness causes uneven reagent penetration, so thicker areas may retain reagent after washing. (Cut paraffin sections at 3–5 μm; 3 μm is recommended.)
4. Folds or bubbles during flotation trap reagent underneath that is hard to wash out. (Gently lift folds or puncture bubbles with forceps.)
5. Poor slide adhesion lets local edges lift, trapping reagent underneath. (Use adhesive slides, or extend baking time.)

2. Procedure

1. Dirty or insufficient dewaxing prevents even antibody penetration. (Use fresh dewaxing reagent or xylene and follow the datasheet dewaxing steps.)
2. Bubbles on the reagent surface during incubation leave uncovered patches. (Puncture bubbles with a pipette tip and gently rock the slide so reagent covers the tissue.)
3. Tissue edges dry from evaporation during incubation and adsorb reagent non-specifically. (Incubate in a humid chamber and gently rock the slide so reagent covers the tissue.)
4. Too few or too gentle distilled-water or PBS washes leave free antibody behind. (Follow the datasheet wash count and time; wet all sections thoroughly.)

F. Whole-slide staining

1. Reagent preparation

1. Excess primary concentration saturates specific signal. (Use the datasheet dilution; verify other ratios with a titration.)
2. Deteriorated DAB has abnormally high catalytic activity. (Prepare DAB fresh and check expiry before use.)

2. Procedure

1. Retrieval that is too long, too high in pH, or too concentrated over-exposes epitopes. (Follow the datasheet retrieval method; verify alternatives experimentally.)
2. Sections dry after retrieval, so dry areas adsorb reagent non-specifically. (Keep sections moist after retrieval; avoid drying.)
3. Sections soak in buffer too long, tissue loosens, and reagent seeps behind the section. (Do not soak sections in buffer for more than 24 h.)
4. Blocking is omitted or incomplete, so endogenous peroxidase reacts directly with DAB. (Immerse sections in 3% H2O2 for 10 min to block endogenous peroxidase.)
5. Incubation that is too long or too hot drives specific binding past saturation. (Incubate at room temperature for the datasheet times.)
6. PBS rinses are skipped during reagent incubations, causing whole-slide non-specific staining. (Follow the datasheet wash count and time; wet all sections thoroughly.)

G. Edge effect

Edge effect is stronger staining at the section margin than in the center.

1. Sample and antigen

1. Uneven fixation after excision leaves uneven target antigen. (Fix large specimens 12–18 h and needle biopsies 4–6 h; adjust to the specimen.)
2. Poor slide adhesion lets local edges lift, trapping reagent underneath. (Use adhesive slides, or extend baking time.)

2. Procedure

1. Retrieval that is too short or too cool opens epitopes unevenly. (Follow the datasheet retrieval method; verify alternatives experimentally.)
2. Bubbles on the reagent surface during incubation leave uncovered patches. (Puncture bubbles with a pipette tip and gently rock the slide so reagent covers the tissue.)
3. Reagent does not fully cover the tissue, so edge reagent evaporates, concentrates, and stains more darkly than the center. (Incubate in a humid chamber and gently rock the slide so reagent covers the tissue.)
4. Slides are not level, so reagent pools to one side and that edge stains more darkly. (Keep slides level during incubation.)
5. Too few or too gentle distilled-water or PBS washes leave antibody in the center. (Follow the datasheet wash count and time; wet all sections thoroughly.)