Preparation of Stained slides of Histological Sections

Introduction
Histology permits microscopic study of tissues. Once the appropriate tissue samples have
been collected they must pass through a series of processes before they can be examined
microscopically. These processes are Fixation, Trimming, Dehydration, Cleaning, Wax
infiltration, Embedding, Sectioning and Staining.

Tissues must be fixed before they can be used for histological observations. Fixative is
used to kill and harden tissues and to preserve cell structures and it will make dissolvable
cell components non-dissolvable. Tissues tend to decompose rapidly after death and
should there fore be fixed quickly to prevent autolysis. If fixative is not at hand, try to
keep the tissue iced or refrigerated. However, the tissue should not be frozen as this will
destroy most cellular details.

Materials
Scalpel Embedding cassettes
Processing cassettes Cold plate or ice tray
Forceps Microtome
Automatic tissue processor and tissue Water bath
embedding agent Staining racks
Paraffin wax Glass slides
Oven or hot plate Cover slips
Pan Compound microscope
Embedding molds

Chemical solutions

Isopropyl alcohol
Chloroform
Xylene (Xylol)
Ethanol
Buffered formalin
Haematoxylin
Eosin
Steps for preparation and Staining of Histological sections

1. Fixation
When processing organs or tissues for histological examination, use a clean scalpel to cut the
tissue into small blocks. The blocks should not be more than 1cm 3 in size. Place them in a
fixative such as Buffered Formalin for 24 hours.

2. Trimming
Trim the blocks or pieces of fixed tissue using a clean scalpel so that they are between 0.2 cm to
0.5cm wide between 0.2 cm to 0.5cm thick and not more than 2.0 cm long. Place trimmed
samples in the labeled cassettes and rinse in tap water for 10 minutes.

3. Dehydration
Carry out dehydration in an automatic tissue processor. Carefully place up to 25 cassettes in
basket number 1 of the processor. If an automatic processor is not available use a series of
staining troughs containing series of isopropyl alcohol as in the following chart.

Timing Schedule for dehydration

Trough Chemical Time duration

No
1 70% Alcohol 3-4 hours
2 90% Alcohol Overnight
3 Absolute Alcohol I Minimum 1 hour
4 Absolute Alcohol II ,, ,,
5 Absolute Alcohol III ,, ,,

4. Cleaning
Place the dehydrated samples in chloroform overnight

5. Wax Infiltration

Wax bath (58 0C) Time duration

1 1 hour
2 1 hour
3 1 hour

6. Embedding sample in wax

Place wax infiltrated samples in embedding molds. Pure melted paraffin and cover with an
embedding cassettes. Pour some more wax on to the cassettes. Place molds on a cold plate or ice
tray so that the embedded samples solidify. After solidification cassettes with wax embedded
samples are separated from the mold and now are ready for sectioning.
7. Sectioning
Remove tissue blocks from mold and trim away excess surrounding paraffin using a clean razor
blade or scalpel. Fit trimmed blocks to a microtome using a pair forceps. Gently operate the
microtome so as it to cut ribbon of section. Put the ribbon to a water bath. Then transfer the
ribbon on to a clean glass slide using a needle. Label slides on the sample holding side with a
glass marking pencil and let them dry on a slide drying bench for about one hour allowing the
sample to hold on to the slide properly.

8. Dewaxing
Place slides in a trough containing xylene (xylol) for 2-3 minutes. Repeat this procedure using
another trough of fresh xylene. Transfer slides through a series of troughs containing descending
grades of alcohol (Absolute alcohol- two times, 95% & 70% for 2-3 minutes each)
Rinse the slides in running tap water for 2-3 minutes and remove any visible artifacts.

9. Staining (Haematoxylin and Eosin)

Method

Earlich’s Haematoxylin 30min

Wash in tap water
Differentiate in 1% acid alcohol dip
“Blue” in tap water or Scott’s Tap Water 5-10min
5% aqueous yellowish eosin 10min
Differentiate in tap water
Dehydrate in absolute alcohol 2min, repeat
Clear in Xylol 3min
Mount in D.P.X

Examine the slides using compound microscope. (Cell nuclei –blue/ cytoplasm, connective
tissues, red blood cells -red or pink.)