Microtechniques 2 - Processing, Embedding

Question 1

What are the 5 steps?

Answer 1

Washing
Dehydration
Clearing agent
Infiltrate in molten paraffin
Embed into moulds

Question 2

What happens during WASHING?

Answer 2

Removing all the fixative. Wash with a buffer/water mix and then rinse again with water to remove any buffer salts. If the fixative is an additive then this is impossible.

Question 3

What happens during DEHYDRATION?

Answer 3

Room temp, 20-30 min each step, intermittent agitation, graded series of ethanol (reduces shrinkage & hardening), all water is replaced.

Question 4

Why is ethanol used instead of methanol, acetone or isoproanol? Methylated spirits?

Answer 4

Those three are more expensive and evaporate readily. Isopropanol the best out of them. Methylated spirits are an acceptable replacement for ethanol.

Question 5

What causes PARCHED EARTH ARTEFACTS?

Answer 5

Improper fixing, when ethanol comes along it attempts to fix the parts that haven’t been fixed properly, it isn’t a good fixative however and therefore PEA occurs.

Question 6

Tissue is stored at 70% concentration ethanol. Why this specific concentration?

Answer 6

Any higher = hardening

Any lower = extraction of tissue components

Question 7

What happens during CLEARING? What are the most common clearing agents used?

Answer 7

Water replaced by a clearing agent (which changes the refractive index). Histoclear/Citroclear most commonly used (comes from limolene, citrus oil).

Question 8

How do we detect tissue that has been cleared by a clearing agent?

Answer 8

It is translucent because the clearing agent results in a change in refractive index.

Question 9

What are alternate clearing agents used?

Answer 9

Xylene: cheap but toxic, preferred over Histoclear because it is so much faster
Toluene: expensive and toxic
Benzene and chloroform: severe health hazard. No longer used!

Question 10

What is a chemical used for COMBINED dehydration and clearing? What are two negatives about it?

Answer 10

Dioxin - carcinogenic, very expensive

Used as a graded series with decreasing amounts of water.

Question 11

What are 8 desirable qualities of an INFILTRATING/EMBEDDING MEDIA? How does paraffin compare?

Answer 11

1. Liquid/solid at a temp that is compatible with the tissue (paraffin melts at 56-60°C)
2. In the liquid phase good penetration of the tissue occurs (paraffin good)
3. No reaction with other tissue components (paraffin not gr8 - dissolves lipids that are poorly fixed)
4. Tissue doesn’t undergo volume change (paraffin not gr8 - always some shrinkage)
5. Good cutting properties (paraffin good - can some mint af ribbons from cutting it)
6. Easily removed from sections before staining (paraffin good - removed with Histoclear)
7. Cheap, readily available
8. Safe - non-toxic, can chuck in regular garbage bin <3

Question 12

What are the 2 types of paraffin wax?

Answer 12

1. Soft wax - 45-50°C - thick sections

2. Hard wax - 55-60°C - thin sections (most commonly used)

Question 13

What are the two types of Automatic Tissue Processors?

Answer 13

1. Tissue transfer

2. Fluid transfer

Question 14

What happens during INFILTRATING?

Answer 14

2-3 changes, 0.5-1hr each, intermittent agitation applied, vacuum applied (speeds it up and makes it more complete).

Question 15

What happens during EMBEDDING?

Answer 15

Poor liquid wax into mould, insert tissue into mould using warm forceps, align tissue into correct position, cool (wax solidifies), label what it is.