Histopathology Slide Preparation (F)

Question 1

What is the process (/ steps) of preparation of tissue for histopathology slides?

Answer 1

1) Fixing tissue
2) Embedding tissue
3) Sectioning the sx
4) Frozen sectioning
5) Basic staining technique

Question 2

What is the purpose of fixation?

Answer 2

To preserve tissue and maintain life-like structure

Question 3

What is the process (/ steps) of fixing tissue?

Answer 3

1) Take fresh or perfused tissue
2) Cut into 1 cm cube
3) Place into fixative (ex. 10% formalin)
4) Leave to fix for 24 - 28 hrs

Question 4

What are the steps in embedding tissue?

Answer 4

1) Preparation
2) Tissue processor
3) Blocking

Question 5

How is preparation (under embedding tissue) done?

Answer 5

After fixation, place into tissue processing cassette

Question 6

How is tissue processor (under embedding tissue) done?

Answer 6

1) The tissue processor automates dehydration, clearing, and infiltration of paraffin wax
2) Dehydration
3) Clearing
4) Infiltration
5) Length of processing is dependent on size and density of tissue: several hrs to overnight

Question 7

How is dehydration done?

Answer 7

Sx is immersed in ascending grades of alcohol to remove H2O and formalin

Question 8

How is clearing done?

Answer 8

An organic solvent (ex. xylene) is used to remove alcohol to allow infiltration w/ paraffin wax

Question 9

How is infiltration done?

Answer 9

Sx is infused w/ molten paraffin wax

Question 10

How is blocking (under embedding tissue) done?

Answer 10

1) Remove cassette containing sx from tissue processor 2) Hold in warming plate until required
3) Fill wax mould w/ wax from dispenser
4) Remove tissue sx from the cassette w/ warmed forceps and place in the mould
5) Place cassette base on top of mould and then fill w/ more wax
6) Place in chilled H2O on cold plate for 30 mins
7) Paraffin wax solidifies to form block containing sx

Question 11

What is the principle of sectioning the sx step?

Answer 11

Produces sections thin enough to allow viewing through a microscope

Question 12

How is sectioning the sx step done?

Answer 12

1) Use rotary microtome to cut tissue sections (3 - 10 um thick)
2) Float tissue section in warm H2O bath to flatten
3) Pick up sections onto glass microscope slide
4) Allow to dry at 37 DC to ensure sections adhere to slide
5) Then, tissue is now ready for staining

Question 13

What is the purpose of frozen sectioning?

Answer 13

It allows rapid dx of fresh tissue

Question 14

How is frozen sectioning done?

Answer 14

1) Freeze sx in cryostat
2) Section frozen sx w/ rotary microtome within freezer cabinet
3) Mount section on warm glass slide (room temp)
4) Dry slide in air
5) Tissue morphology may be compromised but process is quick (ex. for biopsy dx)

Question 15

What is the purpose of the basic staining technique step?

Answer 15

It gives color and contrast to tissue structures

Question 16

What are steps that comprises basic staining technique step?

Answer 16

1) Preparation
2) Staining
3) Dehydrate and mount

Question 17

How is preparation (as a step of basic staining technique step) done?

Answer 17

1) Remove wax w/ a citrus oil based solvent and rehydrate sections through descending alcohols
2) Place sections in tap H2O

Question 18

How is staining (as a step of basic staining technique step) done?

Answer 18

1) Use buffered stain solutions to highlight different structures (ex. hematoxylin stains nuclei blue)
2) Stain tissue w/ hematoxylin (for 10 mins)
3) Rinse slides in tap H2O (between stages)
4) Stain is differentiated by placing in acid-alcohol until the nuclei are stained blue but all other tissue is colorless
5) Neutralize the pH (w/ the use of alkaline tap H2O)
6) Use eosin (for 10 secs) to stain cytoplasm, collagen, and muscle fibers red
7) Between each stage, use a microscope to check for correct lvl of staining

Question 19

How is dehydrate and mount (as a step of basic staining technique step) done?

Answer 19

1) Dehydrate sections w/ ascending grades of alcohol (for 10 mins)
2) Use clearing agent (for 15 mins) to remove all traces of alcohol and raises refractive index to make tissue more transparent
3) Add synthetic mountant (DPX) to a cover slip and then place on top of the section
4) When the adhesive sets, the coverslip will be held in place to protect the stained tissue

Question 20

What are the possible results after doing the staining step?

Answer 20

1) Unstained section
2) Undifferentiated section
3) Differentiated section
4) Section stained w/ eosin only
5) Section stained w/ hematoxylin and eosin