Histology of Endocrine Glands

Comparative Endocrinology
ZOOL-707
Steps
• Extraction of Gland/Tissue
• Fixation of Gland/Tissue
Processing of Gland/Tissue
1. Dehydration
2. Clearing
3. Embedding
4. Sectioning
5. De-waxing
6. Hydration
7. Staining
8. Oiling
9. Clearing
10. Mounting
 Extraction of Gland/Tissue
1. After extraction to remove any blood or
debris attached on the external surface 0.85%
saline solution

8.5g NaCl/1liter water

 Fixation of Gland/Tissue
• In order to preserve cells and tissues constituents in a
condition identical to the existing that life, preventing
autolysis and bacterial decomposition
• Tissues are fixed in Bouin’s fluid and kept in it 6-24
hours depending upon the thickness of the tissues
Bouin’s fluid:
• Saturated Picric acid ( 1g powder/30ml H2O): 75ml
• Formaldehyde: 25ml
• Glacial Acetic acid: 5ml
Picric acid is highly explosive, must be wet form, don’t
touch with metal things as can explode.
 PROCESSING OF TISSUES
• To provide support and a suitable consistency for
microtomy Paraffin Wax method will be used.
1. Dehydration
• Paraffin wax cannot penetrate the tissues in the
presence of water, so dehydration is an essential
preliminary requirement of the process.
• The dehydration is brought about by immersion of
the tissues in the gradual ascending series of alcohol.
Alcohol 70% 90% 100%
Time 1 time 1 time 2 times
Condition Till pale/yellow 4-6 hours 5-6 hours
2. Clearing

• The replacement of alcohol by wax solvent

(e.g., Xylene) is called as clearing.

Xylene 1-Step 2-Step

Time 30 min 15 min
Temperature Room temp 60 ºC in oven
3. Embedding in Wax
• Paper cuvette (box) will be made
• Paraffin wax will be melted
• Melted wax will be poured in the paper cuvette
• Gland/tissue will be placed in wax
• Will be left for hardening
Medium Time Temperature
Paraffin wax 12 hour 60 ◦C in oven at

• Blocks of wax containing gland/tissue will be

stored in fridge
• Wax blocks will be trimmed for extra wax at edges
 4. Sectioning
• The wax block will be attached to a support
• Support will be fixed to microtome
• The thickness of sections will be selected between 7-10µm
• Sections will be cut in the form of ribbon
• Water bath at 42-46ºC will be used for stretching and
wrinkled will be removed and sections will be flattened
• Applying Mayer’s albumin as adhesive substance makes
mounting of section on the glass slide
• The solution is smeared very thinly on the slide before
mounting the section
Mayer’s albumin: Egg white 5 ml
Glycerin 5 ml
Deionized Water 20 µl
 5. De-waxing
• The removal of wax from the sections is called as
“de-waxing”. De-waxing is brought to avoid any
interference during staining.
Two steps
1. The slides are slightly warm on a gentle flame
so the wax begin to dissolve.
2. The de-waxing is carried out by washing in
Xylene.
Medium Repeat Time
Xylene 2 times 5 min each
6. Hydration

• Hydration is brought about to remove the Xylene

from the sections
• The sections are runs through down grades of alcohol
for hydration

Alcohol 100% 90% 70% Tap Water

Repeat 1 time 1 time 1 time 1 time
Time 5 min 5 min 5 min 5 min
7. Staining
• Haematoxylene stains the nucleus
• Eosin stains the cytoplasm
Medium Time
Haematoxylin 10 min
Washing in water 3-5 min
Decolorization Few seconds
(1% HCl in 70% alcohol/ water)
Eosin 1-3 min
Dehydration
Alcohol 90% 100%
Repeat 1 time 1 time
Time 5 min 5 min
8. Oiling

• Clove oil or ceder wood oil is used to give transparency to the

sections for 2-3mins

Medium Time
Clove oil 2-3 min
9. Clearing
• Remove excess of oil in Xylene, which also give transparency
to the section and the removal of alcohol

Xylene 1-Step 2-Step

Repeat 1 time 1 time
Time 2 min 3 min
10. Mounting
• Mounting is carried out to prevent contamination with dust and
air, Canada balsam or D.P.X. (di phenyl Xylene) are used as
mounting agent

Mounting Canada Balsam/ D.P.X. (Di phenyl Xylene)

Microtome