3rd week 2 (embedding & sectioning)

Question 1

Embedding purpose

• Sample embedded in a ….. medium
• gives …. for the tissue structure
• sufficient rigidity to enable ….

Answer 1

• solid medium
• support
• cutting of thin sections

Question 2

Embedding means:

Plastic resins

Paraffin wax

Gelatine and Agarose

soft media&thick section / hard media&thin section / most common

Question 3

aims of preparation and embedding (generally)

Preparation prevents …

Embedding prevents tissue … when cutting

Answer 3

degeneration

distortion

Question 4

difference between OCT and paraffine wax?

Answer 4

commercial, water-soluble embedding material,

paraffine wax not water soluable

Question 5

Steps of embedding:

order and explain

infiltrating / dehydrating / cleaning / embedding

Answer 5

dehydrating / cleaning / infiltrating / embedding

• dehydrating all the water out using alcohol (70%, 90%, 100%)
• cleaning alcohol away by a solvent that mixes with both; alcohol and paraffin wax (xylenes, Histo-clear)
• infiltrating the tissue with molten paraffin wax (replacing xylene/cleaning aid)
• embedding orientating in a metal mould, fill with molten paraffin wax, allow to cool 4 ‘C, followed by microtomy or storing

Question 6

What is a dip and dunk?

From what to what

Answer 6

Tissue processor

graded alcohol -> xylene/Histo-clear -> melted paraffin wax

Question 7

What is an enclosed tissue processor (dip&dunk)?

Answer 7

Uses vacuum to enhance the process

Question 8

After processing [dip&dunk], tissue embedded into paraffin wax station. Steps:

• tissue placed in … …
• …. paraffin wax
• embedding moulds put onto a … ….
• set … block taken out of the mold

Answer 8

• metal molds
• molten
• cold plate
• wax

Question 9

Microtome

Benchtop …. microtome

x-x micron slices

comes out as …

sections are floated onto…

why?

what happens after?

Answer 9

rotary

3-10 micron (micromillimeter)

ribbon

warm water bath (40’C),

expands, sections flatten

mounted on slides (individually or as ribbon)

Question 10

Vibratome

Uses … to cut sections

For softer mediums like:

section thickness:

what happens after?

Answer 10

vibration

agarose, gelatine

50 - 500 micron

sections stained and mounted onto slides

Question 11

Cryostat

Used for snap-frozen tissues, cut at -x’C

Section thickness:

Cut sections can be immediately …. or …

Is embedding needed, why?

Cost of cryostat, cheaper variation?

Answer 11

-20’C

8-50 microns

fixed or stored

Not needed, frozen vitreous water is hard enough for cutting sections, but embedding medium can be used if needed.

Expensive, sliding microtome is a cheaper alternative.

Question 12

Sliding microtome

used for sectioning frozen samples, without expensive cryostat.

Fitted to …

Tissue kept frozen by blasting … or solid …

section thicknes:

Answer 12

benchtop

15 - 200 microns

CO2 or solid CO2 (dry ice, ‘cardice’)