Microtomy and

H&E stain
by Honorable GAKWAYA
 1. Microtomy and paraffin
section preparation
Sectioning of tissue require some skills and
experiences in histotechniques

Standard operating procedures must be

followed
 Process of microtomy and paraffin section preparation

1. Fix specimen properly

 Enhancing the observation of slide
2. process tissue properly
 To allow sectioning properly
 Process con’t

3. Embed specimen carefully

 To allow proper sectioning
4. position microtomy properly
5. Follow safety features properly
 Rules and regulation of using microtome are
followed
 Process con’t

6. Position Blade Clearance Angle Optimally

7. Maintain microtome half life
8. position specimen perfectly
9. Learn about the designing features of your
microtome and how to use them before use.
 Process con’t

10. Take care when trimming , facing or

roughing
11. consider factors affecting section
thickness
12.Make sure that the block is cold
 Process con’t
13. Learn the technique for cutting consistent,
high-quality, thin sections
14. Choose slide and adhesive correctly
15. Float out sections carefully
 Process con’t

16. Dry slide adequately

17. Clean and maintain the microtome
thoroughly
 2. Guidelines for Hematoxylin and Eosin
Staining

One of the stain used in histotechniques,

we have hematoxylin and eosin

All these stain have guideline of how there

are done.
 Reagents

Clearent that are used are xylene and toluene

Steps of clearing
•Clearing by use toluene or xylene
•Deparaffinization by xylene
 Reagent con’t

Alcohol
 Mainly ethanol, denatured ethanol,
isopropyl alcohol.
 Alcohol can dehydrate or hydrate
tissue
 Reagent con’t

Hematoxylin
• This is for deparaffinize, hydrate tissue and
stains the chromatin of cell nucleus.
Bluing Reagent
• It change hematoxylin reddish color to blue
 Reagent con’t

Hematoxylin differentiators like acetate and hydrochloric

acid
Function by removing excess stain and non-specific ones

Tap water, distilled/deionized water

This help to rinse
 Reagent con’t

Eosin (Eosin, eosin-phloxine)

Purpose
This work as counter stain
It stains cells parts that hematoxylin
has not stained especially in cytoplasm
 Rotation and replacement of solutions guideline

Avoid the usage of expired solution.

Wash and rinse each staining vessel.
use the routine of Alcohol & Clearent rotation.
Replace hematoxylin and Eosin should gently
Every day bluing reagent should be prepared
 Record keeping

All daily activities must be reported some

where.
All control of all purchased materials must be
recorded
oDetails of received materials and the date they
have been received
 General comments in preparing/ using
solutions
Optimal staining times should be determined
Room temperature is solution to be used
Have details of all reagent
Labels should be done correctly
All quality controls for reagents must be there
 References

• National Society for Histotechnology. www.nsh.org

• Geoffrey.O.R. ( …). Microtomy and Paraffin Section Preparation.
Scientia Leica Microsystems’ Education Series.
Final!!