Common Problems with H&E Sections and How to Fix Them

 

Histology sectioning rarely goes perfectly the first time. Below are the most frequent issues encountered when cutting and staining H&E sections, along with practical solutions for each.

1. Poor Tissue Fixation

Cause: Tissue that was not properly fixed is dehydrated directly, resulting in blurred, unclear staining.

Solution: After deparaffinizing and rehydrating the section, place it in AFA fixative or acetone for 5 minutes, rinse with water, then proceed with routine staining.

AFA fixative: 850 mL 95% alcohol, 100 mL formalin, 50 mL acetic acid.

2. Holes in the Section

The section shows holes (a sieve-like pattern) and appears broken or uneven.

Solution: This is most common in lymph node and soft tissue samples. Advance the wax block slowly during cutting, trim the tissue gently, and rotate the section several times before lifting it until no holes remain.

Causes of holes:

• Rough trimming done too fast or too aggressively; fine trimming insufficient. Fix: Reduce the feed increment, trim slowly during rough trimming, and only take the section after fine trimming is fully complete.
• Tissue is too hard (e.g., bone with calcification). Fix: Redo decalcification.
• Tissue contains fatty tissue, making it difficult to cut. Fix: Adjust section thickness.

3. Uneven Section Thickness

The section is uneven in thickness or shows ridges, producing a "terraced" appearance after staining.

Causes:

1. Damaged blade holder, leaving a gap between blade and blade seat
2. Foreign matter between the clamp plate and blade holder
3. Cutting too fast or unevenly on harder tissue (e.g., uterus)
4. Embedding cassette too small, poor clamping

Solutions:

1. Replace the blade holder
2. Clean any foreign matter between the clamp plate and blade holder
3. For harder tissue, warm the wax block gently with your fingers, trim with small increments, complete fine trimming fully, then cut slowly and evenly
4. Place the embedding cassette horizontally in the clamp for sectioning

4. Tissue Wrinkling

Common causes:

1. Wax block not sufficiently cold
2. Blade overused and dulled
3. Wax residue on the blade edge dulling it
4. Embedding wax too hard
5. Blade holder arc too large

Solutions:

1. Re-freeze the wax block
2. Reposition or replace the blade
3. Gently wipe the blade along the cutting edge with damp gauze
4. Switch to a lower-melting-point wax
5. Reduce the blade holder's arc
6. Slightly raise the flotation bath temperature

5. Incomplete Sections — Under-cut or Over-cut

Cause: In single or multi-block embedding, the tissue block or cassette bottom surface is not level.

Solution: Adjust the angle of the universal cassette clamp, or re-embed the tissue.

6. Sections Won't Form a Continuous Ribbon

Cause: Wax block not cold enough; embedding wax too hard or too soft.

Solution: Replace the wax and re-embed.

7. Sections Compress or Pile Up

Cause: Wax block not cold enough; embedding wax too viscous; static electricity.

Solution: Cut as thin as possible, and use a humidifier during sectioning.

8. Wax Ribbon Curves Into a Fan Shape

Causes:

1. Irregular wax block shape
2. Uneven blade sharpness — one side sharp, the other dull

Solutions:

1. Change the blade position, trim the wax block, and re-cut
2. Replace with a new blade

9. Section Contamination

Common sources:

1. Tissue debris contamination — fragments from other tissue stick to the ribbon and get picked up. Fix: Brush the cutting stage clean thoroughly during fine trimming.
2. Water surface contamination — an unclean water bath picks up nearly invisible debris when floating the section. Fix: Skim the water surface frequently with absorbent paper to keep the flotation bath clean.
3. Contamination during baking — dust falls onto the tissue, or overly forceful brushing sends debris flying onto the slide. Fix: Brush gently, and re-cut the section if visible contamination occurs.
4. Contamination from stains/reagents — pigment particles or reagent sediment adhere to the section during staining. Fix: Filter staining solutions regularly and replace reagents often.

10. Section Curls Instead of Forming a Ribbon

Causes:

1. Blade not sharp
2. Blade angle too large
3. Section too thick
4. Wax block not cold enough

Solutions:

1. Replace with a new blade
2. Adjust the blade angle (5°)
3. Section thickness is generally 3–5 μm, varying by tissue type: kidney 1–2 μm, lymph node 2–3 μm, brain 4–5 μm, adipose tissue 6–7 μm
4. Increase the wax block's freezing time

11. Section Sticking

Cause: High room temperature or dry air increases static electricity during sectioning, causing the section to stick to the blade and preventing ribbon formation.

Solution: Use a humidifier if possible during sectioning, and re-freeze the wax block.

12. Tissue Edge Curling

Cause: Common in harder tissues such as uterus, prostate, and poorly decalcified samples.

Solution: Use anti-detachment solution or egg white/glycerin when mounting the section, drain excess water before baking, and avoid excessive force when rinsing during staining.