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QIHC practice questions

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Question 1Preanalytic variables and specimen handling (fixation, processing, sectioning, antigen preservation)

Which fixative is the standard for routine formalin-fixed, paraffin-embedded tissue intended for immunohistochemistry?

  1. 10% neutral buffered formalin Correct answer.
  2. Absolute ethanol dehydration
  3. Zenker's mercuric chloride fixative
  4. Bouin's picric acid fixative

Explanation

10% neutral buffered formalin (approximately 4% formaldehyde) is the routine fixative for FFPE tissue because it preserves morphology and antigens when fixation is properly controlled. Other fixatives may preserve selected antigens but require special processing or retrieval.

Question 2Preanalytic variables and specimen handling (fixation, processing, sectioning, antigen preservation)

What section thickness is generally recommended for immunohistochemical staining of paraffin sections?

  1. 15–20 µm
  2. 1–2 µm
  3. 8–10 µm
  4. 3–5 µm Correct answer.

Explanation

Sections of about 3–5 µm are thin enough for reagent penetration and morphologic detail while retaining sufficient tissue for staining. Thicker sections can trap reagents and increase background.

Question 3Preanalytic variables and specimen handling (fixation, processing, sectioning, antigen preservation)

Why are paraffin sections typically baked at about 60°C before immunohistochemical staining?

  1. To activate masked tissue antigens
  2. To dissolve residual paraffin wax
  3. To improve section adhesion Correct answer.
  4. To quench endogenous peroxidase

Explanation

Baking melts surface paraffin and helps the section adhere firmly to the slide, reducing detachment during heat retrieval and washing. Dewaxing and antigen retrieval are separate steps.

Question 4Preanalytic variables and specimen handling (fixation, processing, sectioning, antigen preservation)

Which slide surface is most appropriate for IHC sections that will undergo heat-induced antigen retrieval?

  1. Charged silane-coated slides Correct answer.
  2. Glass slides with coverslips attached
  3. Plain uncoated glass slides
  4. Frosted-end glass slides

Explanation

Heat-induced retrieval and repeated washes can detach sections from plain glass. Charged slides, such as silane- or poly-L-lysine-coated slides, improve adhesion.

Question 5Preanalytic variables and specimen handling (fixation, processing, sectioning, antigen preservation)

Prolonged fixation in neutral buffered formalin can produce which artifact in IHC?

  1. Loss of tissue morphology and architecture
  2. Increased endogenous peroxidase activity
  3. Complete removal of paraffin wax
  4. Epitope masking by cross-linking Correct answer.

Explanation

Excessive formalin fixation increases protein cross-linking, which can mask epitopes and produce weak or absent staining. The effect may require stronger antigen retrieval or be irreversible.

Question 6Preanalytic variables and specimen handling (fixation, processing, sectioning, antigen preservation)

Which decalcifying agent best preserves antigens for IHC of bone marrow core biopsies?

  1. Hydrochloric acid
  2. Nitric acid
  3. EDTA Correct answer.
  4. Formic acid

Explanation

EDTA is a chelating decalcifier that removes calcium slowly and is gentler on antigens and nucleic acids than strong acids. Acid decalcifiers can accelerate antigen loss.

Question 7Preanalytic variables and specimen handling (fixation, processing, sectioning, antigen preservation)

Cold ischemia time refers to which interval in specimen handling?

  1. From fixation to processing
  2. From embedding to sectioning
  3. From sectioning to staining
  4. From tissue excision to fixation Correct answer.

Explanation

Cold ischemia time is the period between removal of tissue from the body and placement in fixative. Prolonged cold ischemia allows autolysis and can degrade labile antigens.

Question 8Preanalytic variables and specimen handling (fixation, processing, sectioning, antigen preservation)

How should unstained paraffin sections be stored before IHC to minimize antigen loss?

  1. At 4°C Correct answer.
  2. At 37°C
  3. At room temperature in light
  4. Immersed in formalin

Explanation

Unstained sections are best stored refrigerated at about 4°C and stained promptly, because antigens can deteriorate over time at warmer temperatures. Light exposure is also minimized.

Question 9Preanalytic variables and specimen handling (fixation, processing, sectioning, antigen preservation)

What pH range is recommended for 10% neutral buffered formalin used in IHC?

  1. 3.0–4.0
  2. 8.5–9.5
  3. 10.0–11.0
  4. 6.8–7.4 Correct answer.

Explanation

Neutral buffered formalin is buffered near physiologic pH, approximately 6.8–7.4, to limit acid-induced antigen alteration. Acidic or strongly alkaline formalin can harm morphology and antigenicity.

Question 10Preanalytic variables and specimen handling (fixation, processing, sectioning, antigen preservation)

A paraffin section cut at 8 µm instead of 4 µm for IHC is most likely to cause which problem?

  1. Inability to counterstain nuclei
  2. Failure of paraffin dewaxing
  3. Complete loss of all antigenicity
  4. Poor penetration and background Correct answer.

Explanation

Thick sections can impede uniform reagent penetration and trap reagents, leading to weak central staining and increased background. A 3–5 µm section is preferred.

Question 11Preanalytic variables and specimen handling (fixation, processing, sectioning, antigen preservation)

Why should strong acid decalcification be avoided before IHC?

  1. It denatures antigens and DNA Correct answer.
  2. It raises the paraffin melting point
  3. It causes excessive tissue adhesion
  4. It blocks endogenous peroxidase

Explanation

Strong acids can denature proteins and nucleic acids, producing false-negative IHC and poor molecular results. EDTA or mild acid decalcification is preferred when antigen preservation matters.

Question 12Preanalytic variables and specimen handling (fixation, processing, sectioning, antigen preservation)

Which antigen retrieval approach is appropriate for a nuclear antigen that requires high-pH conditions?

  1. Protease digestion at 37°C
  2. Heat-induced retrieval with EDTA pH 9 Correct answer.
  3. Acid citrate buffer at pH 6 only
  4. No antigen retrieval at all

Explanation

High-pH heat-induced retrieval, commonly with EDTA buffer at pH 9, is used for many nuclear epitopes that are poorly unmasked at acidic pH. The optimal method must be validated for each antibody.

Question 13Preanalytic variables and specimen handling (fixation, processing, sectioning, antigen preservation)

When heat-induced antigen retrieval fails to unmask an epitope, which enzymatic alternative is commonly used?

  1. DNase digestion
  2. Protease digestion Correct answer.
  3. Lipase digestion
  4. Amylase digestion

Explanation

Proteases such as proteinase K, trypsin, or pepsin can digest cross-linked proteins and expose some epitopes that resist heat retrieval. Overdigestion can destroy morphology and antigen.

Question 14Preanalytic variables and specimen handling (fixation, processing, sectioning, antigen preservation)

Compared with formalin-fixed tissue, alcohol-fixed tissue for IHC generally shows what?

  1. Complete loss of all antigens
  2. Better preservation of some labile antigens Correct answer.
  3. Universal stronger staining for all antibodies
  4. No need for antigen retrieval

Explanation

Alcohol fixation precipitates proteins and can better preserve certain labile antigens, but it produces different morphology and is not universally superior. Retrieval requirements vary by antibody.

Question 15Preanalytic variables and specimen handling (fixation, processing, sectioning, antigen preservation)

What is the recommended fixative for a cytology cell block intended for IHC?

  1. Distilled water with saline
  2. Acetone dehydration only
  3. 10% buffered formalin Correct answer.
  4. Xylene clearing solution

Explanation

Cell blocks are usually fixed in 10% neutral buffered formalin so that they can be processed and stained like paraffin tissue sections. Direct smears may instead be air-dried or alcohol-fixed.

Question 16Preanalytic variables and specimen handling (fixation, processing, sectioning, antigen preservation)

Delayed fixation of a resection specimen most directly causes which preanalytic problem?

  1. Overdecalcification of bone
  2. Autolysis and antigen degradation Correct answer.
  3. Excessive formalin cross-linking
  4. Incomplete paraffin infiltration

Explanation

Ischemia before fixation allows endogenous enzymes to degrade cells and proteins, which can weaken or alter IHC staining. Cold ischemia time should therefore be minimized.

Question 17Preanalytic variables and specimen handling (fixation, processing, sectioning, antigen preservation)

Why may fatty tissue such as breast adipose require longer fixation?

  1. Fat dissolves in formalin
  2. Fat cells contain more antigen
  3. Adipose tissue is hypertonic
  4. Lipid impedes fixative penetration Correct answer.

Explanation

Adipose tissue is poorly penetrated by aqueous fixatives because lipid is hydrophobic, so thicker fatty specimens need more time or sectioning to ensure adequate fixation. Inadequate fixation can cause weak staining and poor morphology.

Question 18Preanalytic variables and specimen handling (fixation, processing, sectioning, antigen preservation)

Prolonged drying of sections at high temperature before IHC can cause what?

  1. Improved adhesion
  2. Complete dewaxing
  3. Antigen loss Correct answer.
  4. Better morphology

Explanation

Excessive heat and drying can denature or degrade antigens and increase background, so baking should be controlled. Gentle drying or short baking is preferred.

Question 19Preanalytic variables and specimen handling (fixation, processing, sectioning, antigen preservation)

Why is a positive control tissue often placed on the same slide as the patient section?

  1. To increase antigen retrieval
  2. To prevent tissue detachment
  3. To verify reagents and retrieval Correct answer.
  4. To quench endogenous peroxidase

Explanation

An on-slide positive control demonstrates that fixation, retrieval, reagents, and detection worked for that run. If the control fails, patient staining cannot be interpreted as negative.

Question 20Preanalytic variables and specimen handling (fixation, processing, sectioning, antigen preservation)

Which storage condition best preserves antigenicity in paraffin blocks long term?

  1. Cool, dry, dark environment Correct answer.
  2. Immersion in formalin
  3. Warm, humid environment
  4. Freezing at -20°C

Explanation

Paraffin blocks are best kept cool, dry, and protected from light; heat and humidity can degrade antigens and promote mold. Formalin immersion would damage the block.

Question 21Preanalytic variables and specimen handling (fixation, processing, sectioning, antigen preservation)

For HER2 IHC in breast cancer, which preanalytic variables are specifically regulated because they can alter results?

  1. Cold ischemia and fixation time Correct answer.
  2. Slide charge and drying time
  3. Counterstain color and coverslip
  4. Section thickness and slide charge

Explanation

HER2 testing guidelines specify limits for time to fixation and duration of formalin fixation because both underfixation and overfixation can change HER2 IHC results. Ischemia and fixation are critical preanalytic variables.

Question 22Preanalytic variables and specimen handling (fixation, processing, sectioning, antigen preservation)

A tissue section shows strong IHC staining at the edge but weak staining in the center. Which preanalytic cause is most likely?

  1. Poor central fixative penetration Correct answer.
  2. Section cut too thin
  3. Over-decalcification of tissue
  4. Excessive antigen retrieval

Explanation

Edge-positive, center-negative staining suggests inadequate fixative diffusion into the center, leaving central antigens underfixed or degraded. Larger specimens should be sliced to allow fixative penetration.

Question 23Preanalytic variables and specimen handling (fixation, processing, sectioning, antigen preservation)

Which statement best describes the relationship between antigen retrieval pH and epitope detection?

  1. The optimal pH varies by epitope Correct answer.
  2. pH 6 is universally optimal
  3. pH 9 always destroys antigens
  4. pH has no effect on staining

Explanation

Different epitopes require different retrieval conditions; some work best at acidic pH and others at alkaline pH. Antibody validation should include the appropriate retrieval solution.

Question 24Preanalytic variables and specimen handling (fixation, processing, sectioning, antigen preservation)

Bouin's fixative can interfere with IHC primarily because it does what?

  1. Masks antigens and imparts yellow color Correct answer.
  2. Activates endogenous peroxidase
  3. Overdecalcifies mineralized tissue
  4. Increases the paraffin melting point

Explanation

Bouin's solution contains picric acid, which can mask antigens and leave a yellow discoloration that must be removed before interpretation. Special washing or retrieval may be needed.

Question 25Preanalytic variables and specimen handling (fixation, processing, sectioning, antigen preservation)

In bone marrow biopsies, over-decalcification with acid is most likely to produce which IHC result?

  1. Improved nuclear staining intensity
  2. No effect on IHC results
  3. False-positive staining from more antigen
  4. False-negative staining from antigen loss Correct answer.

Explanation

Excessive acid exposure destroys antigens and nucleic acids, leading to weak or negative staining that does not reflect the true expression. EDTA decalcification minimizes this risk.

Question 26Preanalytic variables and specimen handling (fixation, processing, sectioning, antigen preservation)

When trimming a solid tissue specimen before immersion in 10% neutral buffered formalin for later IHC, the slices should be cut at approximately what thickness?

  1. 1–2 mm
  2. 10–15 mm
  3. 3–5 mm Correct answer.
  4. 20–25 mm

Explanation

Fixative penetrates tissue by diffusion at roughly 1 mm per hour from the surface, so slices must be thin enough for the fixative to reach the center in a reasonable time. Slices of about 3–5 mm are the standard compromise between adequate penetration and practical handling.

Question 27Preanalytic variables and specimen handling (fixation, processing, sectioning, antigen preservation)

What is the main reason sections for IHC are mounted on charged or adhesive-coated slides?

  1. To block endogenous peroxidase activity
  2. To flatten wrinkles during drying
  3. To prevent the section detaching during heated retrieval Correct answer.
  4. To increase antibody penetration into the section

Explanation

Heat-induced antigen retrieval exposes sections to near-boiling buffer for extended periods, which can lift sections off untreated glass. Electrostatic or adhesive coatings anchor the section so it survives retrieval and repeated washing.

Question 28Preanalytic variables and specimen handling (fixation, processing, sectioning, antigen preservation)

A 2 cm³ piece of tumor is submitted for IHC in a container of 10% neutral buffered formalin. What is the minimum volume of fixative that should accompany it?

  1. 2 mL
  2. 6 mL
  3. 20 mL Correct answer.
  4. 10 mL

Explanation

The accepted minimum fixative-to-tissue ratio is 10:1 by volume, so a 2 cm³ specimen requires at least 20 mL of formalin. Falling below this ratio depletes and dilutes the fixative, slowing fixation and allowing antigen degradation.

Question 29Preanalytic variables and specimen handling (fixation, processing, sectioning, antigen preservation)

A section shows uniformly weak staining across the entire patient tissue while the on-slide positive control stains as expected. Records show the specimen sat in saline at room temperature overnight before being placed in formalin. Which preanalytic event best explains the finding?

  1. Formalin pigment masking the epitope
  2. Autolysis degrading the target epitope before fixation Correct answer.
  3. Incomplete antigen retrieval affecting only the patient tissue
  4. Over-decalcification of the specimen

Explanation

Unfixed tissue held in a non-fixative medium activates endogenous enzymes that digest proteins, so epitopes can be lost before cross-linking begins. Because the control tissue was handled correctly and stained appropriately, the defect lies in the patient specimen's pre-fixation handling.

Question 30Preanalytic variables and specimen handling (fixation, processing, sectioning, antigen preservation)

A section shows abundant brown granular deposit scattered over all tissue, including regions expected to be negative, and the deposit is still present in the negative reagent control. The specimen was fixed in a container of formalin that had stood open for months. What is the most likely cause?

  1. Endogenous peroxidase activity
  2. Formalin pigment from acidic fixative Correct answer.
  3. Insufficient DAB incubation
  4. Excessive antigen retrieval

Explanation

Formalin that is unbuffered or stored open becomes acidic, and acid formaldehyde reacts with hemoglobin to form acid hematin (formalin pigment), a brown granular deposit. Because it is inherent to the tissue, it persists in negative controls and can mimic DAB; it can be removed with saturated alcoholic picric acid.

Question 31Antigen retrieval and pretreatment (heat-induced and enzymatic methods, buffer selection, optimization)

What is the primary purpose of antigen retrieval in immunohistochemistry on formalin-fixed, paraffin-embedded tissue?

  1. To remove endogenous peroxidase activity from the tissue
  2. To restore epitopes that were masked by formalin fixation Correct answer.
  3. To increase the concentration of antibody applied to the section
  4. To counterstain nuclei before microscopic evaluation

Explanation

Formalin fixation creates methylene cross-links between proteins that sterically hide many epitopes; retrieval breaks or loosens these cross-links so antibody can bind. Antibody concentration, peroxidase blocking, and counterstaining are separate steps of the immunohistochemical workflow.

Question 32Antigen retrieval and pretreatment (heat-induced and enzymatic methods, buffer selection, optimization)

Which buffer is the classic heat-induced epitope retrieval solution at pH 6.0?

  1. Citrate, pH 6.0 Correct answer.
  2. Glycine-HCl, pH 2.2
  3. Phosphate-buffered saline, pH 7.4
  4. Tris-HCl, pH 9.5

Explanation

Citrate buffer at 10 mM, pH 6.0 is the most widely used acidic HIER solution and remains a standard first choice for many antibodies. The other buffers listed serve different purposes or pH ranges in the laboratory.

Question 33Antigen retrieval and pretreatment (heat-induced and enzymatic methods, buffer selection, optimization)

What is the main consequence of excessive heat-induced antigen retrieval?

  1. Irreversible inactivation of endogenous alkaline phosphatase
  2. Loss of tissue morphology and possible false-negative staining Correct answer.
  3. Stronger specific staining with no adverse effect
  4. Selective enhancement of nuclear staining only

Explanation

Over-heating or prolonged boiling, particularly at alkaline pH, can denature or extract the target protein and cause sections to detach, producing weak staining with degraded morphology. Retrieval must therefore be optimized for time, temperature, and pH rather than maximized.

Question 34Antigen retrieval and pretreatment (heat-induced and enzymatic methods, buffer selection, optimization)

Enzymatic antigen retrieval with trypsin is conventionally performed at which temperature?

  1. 4 °C
  2. 95 °C
  3. 37 °C Correct answer.
  4. 25 °C

Explanation

Protease digestion is carried out at 37 °C in a humid chamber because the enzymes are active at physiological temperature and are denatured or inactivated by heat. Digestion time and enzyme concentration are the variables adjusted during optimization.

Question 35Antigen retrieval and pretreatment (heat-induced and enzymatic methods, buffer selection, optimization)

Why do many epitopes become undetectable after formalin fixation unless retrieval is performed?

  1. Formalin completely degrades proteins into small peptides
  2. Formalin forms methylene cross-links that mask antigenic sites Correct answer.
  3. Formalin only increases tissue autofluorescence
  4. Formalin dissolves membrane lipids and removes the antigen

Explanation

Formaldehyde reacts with amino groups to create methylene bridges between adjacent proteins, which sterically hide epitopes from the antibody. Retrieval reverses or loosens these cross-links to re-expose the antigenic sites.

Question 36Antigen retrieval and pretreatment (heat-induced and enzymatic methods, buffer selection, optimization)

Heat-induced epitope retrieval is typically performed in which temperature range?

  1. 4–10 °C
  2. 95–100 °C Correct answer.
  3. 55–65 °C
  4. 37–45 °C

Explanation

HIER relies on near-boiling buffer, usually 95–100 °C, delivered by microwave, water bath, steamer, or pressure cooker. Lower temperatures do not efficiently break formalin cross-links.

Question 37Antigen retrieval and pretreatment (heat-induced and enzymatic methods, buffer selection, optimization)

What should be done immediately after heat-induced retrieval and before applying the primary antibody?

  1. Apply the primary antibody while the slides are still hot
  2. Block with serum at 60 °C
  3. Cool the slides in buffer and rinse Correct answer.
  4. Rinse the slides in distilled water only

Explanation

Slides are allowed to cool, usually while still immersed in the retrieval buffer, and are then rinsed before blocking and primary antibody incubation. Applying antibody to hot slides gives uneven staining and increases the risk of section detachment.

Question 38Antigen retrieval and pretreatment (heat-induced and enzymatic methods, buffer selection, optimization)

Which reagent is used to block endogenous peroxidase activity before peroxidase-based detection?

  1. Hydrogen peroxide Correct answer.
  2. Sodium azide
  3. Normal goat serum
  4. Bovine serum albumin

Explanation

Hydrogen peroxide, typically 3%, inactivates endogenous peroxidases that would otherwise generate nonspecific chromogen signal. Serum and albumin are protein blocks against nonspecific binding, and azide inhibits peroxidase rather than blocking tissue enzyme.

Question 39Antigen retrieval and pretreatment (heat-induced and enzymatic methods, buffer selection, optimization)

Why is EDTA included in alkaline antigen retrieval buffers?

  1. It coats the section and reduces hydrophobic background
  2. It raises the pH above 12 without added base
  3. It chelates divalent cations and destabilizes calcium-dependent protein complexes Correct answer.
  4. It digests peptide bonds in cross-linked proteins

Explanation

EDTA sequesters calcium and magnesium, which weakens calcium-stabilized protein interactions and helps expose epitopes at high pH. It does not cleave proteins; that is the action of proteolytic enzymes.

Question 40Antigen retrieval and pretreatment (heat-induced and enzymatic methods, buffer selection, optimization)

A standard citrate antigen retrieval working solution is prepared at which concentration and pH?

  1. 50 mM, pH 7.4
  2. 100 mM, pH 8.0
  3. 10 mM, pH 6.0 Correct answer.
  4. 1 M, pH 4.0

Explanation

The classic citrate HIER buffer is 10 mM citric acid and sodium citrate adjusted to pH 6.0. Higher molarity or different pH values define other retrieval systems with different stringency.

Question 41Antigen retrieval and pretreatment (heat-induced and enzymatic methods, buffer selection, optimization)

A nuclear antigen stains weakly with citrate pH 6.0 retrieval, and the antibody is known to perform well. What is the most reasonable next step?

  1. Shorten the retrieval time by half
  2. Lower the retrieval pH to 4.0
  3. Increase the primary antibody dilution
  4. Switch to Tris-EDTA buffer at pH 9.0 Correct answer.

Explanation

Nuclear antigens frequently require more stringent retrieval, and moving from acidic citrate to alkaline Tris-EDTA is the standard next step in optimization. Lowering pH or shortening time would reduce retrieval further, and greater dilution reduces signal.

Question 42Antigen retrieval and pretreatment (heat-induced and enzymatic methods, buffer selection, optimization)

Which change is most characteristic of excessive enzymatic digestion?

  1. Sharper nuclear detail
  2. No measurable effect on the section
  3. Stronger staining with clean background
  4. Loss of tissue architecture with weak or absent staining Correct answer.

Explanation

Over-digestion degrades tissue structure and can destroy the epitope itself, so morphology deteriorates while specific signal falls. The aim is the shortest digestion that gives maximal specific signal with preserved architecture.

Question 43Antigen retrieval and pretreatment (heat-induced and enzymatic methods, buffer selection, optimization)

What pretreatment is generally needed for immunohistochemistry on fresh frozen sections?

  1. No antigen retrieval is usually necessary Correct answer.
  2. Proteinase K digestion for 30 minutes
  3. Trypsin digestion at 37 °C for 1 hour
  4. Strong heat retrieval at pH 9.0 for 40 minutes

Explanation

Frozen sections have not been exposed to formalin cross-linking, so epitopes are generally accessible without retrieval; a brief fixation before staining is common. Aggressive heat or protease treatment mainly risks damaging the section.

Question 44Antigen retrieval and pretreatment (heat-induced and enzymatic methods, buffer selection, optimization)

When a protocol requires both heat-induced and enzymatic retrieval, in what order are they usually applied?

  1. Either order works equally well with no effect
  2. Heat retrieval first, then enzymatic digestion Correct answer.
  3. Enzymatic digestion first, then heat retrieval
  4. Both simultaneously in the same buffer

Explanation

Heat-induced retrieval is performed first because subsequent heating would denature and inactivate the protease. The enzyme is then applied at 37 °C for a controlled time before staining.

Question 45Antigen retrieval and pretreatment (heat-induced and enzymatic methods, buffer selection, optimization)

Why are slides allowed to cool gradually in the retrieval buffer after heating?

  1. Cooling supports epitope re-folding and limits section detachment Correct answer.
  2. Cooling raises the buffer pH to 9.0
  3. Cooling inactivates endogenous peroxidase
  4. Cooling chemically cleaves residual cross-links

Explanation

Gradual cooling lets proteins settle into a conformation the antibody can recognize and avoids thermal shock that can lift sections off the glass. Peroxidase blocking, buffer pH, and cross-link cleavage are not achieved by cooling.

Question 46Antigen retrieval and pretreatment (heat-induced and enzymatic methods, buffer selection, optimization)

What is the best way to establish optimal retrieval conditions for a newly introduced antibody?

  1. Copy the protocol used for another antibody on the same section
  2. Use the longest retrieval time available in the laboratory
  3. Test a matrix of pH, temperature, and time on appropriate control tissue Correct answer.
  4. Rely entirely on the manufacturer's suggested protocol

Explanation

Optimal retrieval is antibody-specific and is established empirically by comparing results across pH, temperature, and time on known positive and negative control tissue. Published protocols are starting points that still require local verification.

Question 47Antigen retrieval and pretreatment (heat-induced and enzymatic methods, buffer selection, optimization)

Prolonged high-temperature retrieval can reduce staining for some phospho-specific antibodies because:

  1. Heat converts the buffer to a strongly acidic pH
  2. Heat increases phosphatase activity within the antibody solution
  3. Prolonged heating can promote dephosphorylation of the target residue Correct answer.
  4. Heat removes phosphate ions from the retrieval buffer

Explanation

Phosphorylated residues are heat-labile and tissue phosphatases remain active during prolonged heating, so the epitope can lose its phosphate group. Reducing retrieval time and temperature, or adjusting the buffer, preserves the phospho-epitope.

Question 48Antigen retrieval and pretreatment (heat-induced and enzymatic methods, buffer selection, optimization)

A commonly used Tris-EDTA antigen retrieval buffer at pH 9.0 contains EDTA at approximately what concentration?

  1. 1 mM Correct answer.
  2. 100 mM
  3. 500 mM
  4. 10% (w/v)

Explanation

The standard Tris-EDTA retrieval buffer is about 10 mM Tris with 1 mM EDTA adjusted to pH 9.0. Millimolar EDTA is sufficient to chelate divalent cations without stripping the tissue.

Question 49Antigen retrieval and pretreatment (heat-induced and enzymatic methods, buffer selection, optimization)

Why might switching a well-staining membrane antigen from citrate pH 6.0 to a long pH 9.0 heat protocol abolish the signal?

  1. Excessive heat at alkaline pH can denature or extract the target protein Correct answer.
  2. EDTA binds and sequesters the primary antibody
  3. Alkaline pH converts endogenous peroxidase into a blocking agent
  4. Alkaline pH directly blocks the antibody's antigen-binding site

Explanation

Over-retrieval, especially under harsher alkaline conditions, can unfold or solubilize the antigen so no intact target remains for the antibody to bind. Retrieval should be tuned to each antigen rather than maximized.

Question 50Antigen retrieval and pretreatment (heat-induced and enzymatic methods, buffer selection, optimization)

Tissue that has been fixed in formalin for several days is likely to need which retrieval adjustment?

  1. Longer retrieval at an alkaline pH, validated against controls Correct answer.
  2. Shorter retrieval time than usual
  3. Enzymatic digestion only, with no heating
  4. No retrieval at all

Explanation

Extended fixation creates more methylene cross-links, so additional retrieval energy is usually required, often at pH 9.0 with longer times. Controls confirm that the stronger protocol exposes the epitope without destroying morphology.