PRELIM: CH6 Fixation (2)

Surface drying and permanent damage

Small biopsies are especially prone to:

≤5 mm (≤4 mm preferred)

Maximum recommended tissue thickness for routine fixation is:

Cut thinner and fix longer

The best approach for fatty tissues is to:

Cover with layers of gauze to hold it under

To keep an air-filled lung submerged during fixation:

Suspend whole in 10% buffered formalin for 2–3 weeks

For brains, recommended fixation before gross sectioning is:

Collapse and wrinkling occur due to vitreous escape

Eyes should not be dissected before fixation primarily because:

Stretch with sutures or lay flat on moist paper before fixation

For muscle biopsies to avoid contraction artifacts, it is recommended to:

Water (risk of glycogen dissolution)

Glycogen-containing tissues should avoid:

Fixative penetration is limited and slower than surface reaction

The interior of a large, thick specimen often remains under-fixed because:

Artifact pigments remaining in sections

Incomplete washing after fixation can lead to:

Partial coagulation by ethanol and incomplete wax impregnation

“Crush artifact” in liver biopsies can be associated with:

Frozen (cryostat) sections with lipid stains (e.g., Oil Red O)

For demonstrating lipids histochemically in routine pathology, use:

Aldehydes (due to amino groups)

Phospholipids are generally fixed by:

Formaldehyde reacts with unsaturated fatty acids

Long storage in formaldehyde reduces lipid demonstration because:

Alcohol-based fixatives (e.g., cold absolute alcohol, Rossman’s)

For glycogen retention, a superior approach is:

Neutral buffered formal saline/formaldehyde vapor

The most common fixative for protein histochemistry is:

Glutaraldehyde, paraformaldehyde, osmium tetroxide

The preferred primary fixatives for EM include:

Karnovsky’s paraformaldehyde–glutaraldehyde

For EM, a widely used combined fixative is:

Preserve enzyme activity and localization, often with mild fixation or frozen sections

For enzyme histochemistry, the ideal is to:

Frozen sections rather than formalin-fixed paraffin-embedded tissue

In immunofluorescence (IF), to minimize autofluorescence, it is best to use:

Autofluorescence under fluorescence microscopy

Formalin-fixed paraffin-embedded tissues frequently exhibit:

A few seconds to minutes in cold acetone or methanol

For Immunofluorescence on sensitive antigens, fixation can be limited to:

Continued cross-linking reducing antigenicity

Over-fixation in formalin impacts IHC (immunohistochemistry) by:

Antigen retrieval (HIER or enzyme digestion)

To restore antigenicity in IHC after cross-linking, labs commonly use:

Boiling in pH 6 citrate buffer ~10 minutes with additional hot incubation

HIER (Heat-induced epitope retrieval) using microwave typically involves:

pH 3 citrate or pH 8–9 Tris-EDTA

In some cases, alternative HIER (Heat-induced epitope retrieval) buffers use:

Trypsin, protease, or pepsin with optimized time/concentration

Enzyme-mediated retrieval can employ:

Do not reuse diluted antibodies due to adsorption losses

A practical note for antibody handling:

Destroy antibody activity

Freeze/thaw cycles of antibodies:

Subsequent permeabilization

Methanol fixation at −20°C typically requires:

Often obviates the need for separate permeabilization

Acetone fixation at −20°C for 5–10 minutes:

Permeabilization using 0.5% Triton X-100

Formalin fixation for cultured cells (IF/IHC) often follows with:

Preserve structure by cross-linking and permit antibody access

Paraformaldehyde (PFA) fixation with Triton is used to:

Zonal fixation: outer rim over-fixed, center under-fixed

The principal risk of using hot fixative on thick blocks is:

Heat that accelerates diffusion and chemical reactions

Microwave fixation’s major mechanism is: