Histological Staining of rat tissues
Introduction:
Cellular components differ in their absorption of light and most tissue sections are invisible
using ordinary light microscopes - therefore, histological stains are used to treat the sections
that bind selectively to different structures according to their charges, making them different
colours. During this practical, the main aim was to visualise the muscle, lung and kidney rat
tissues using Hematoxylin and Eosin stain (H&E). Hematoxylin is a basic dye that binds to
negatively charged structures such as DNA and RNA due to negatively charged phosphate
groups in the sugar-phosphate backbone, and makes them appear violet under the microscope.
On the other hand, Eosin stain binds to positively charged substances such as amino acids and
proteins present in muscle fibres and cytoplasmic filaments, making them appear pink/red.
Hematoxylin and Trichrome stain was used to visualise the kidney tissue.
Materials and method:
Hematoxylin and Eosin (H&E): For lung and muscle tissues
1. Stain in Harris Alum Haematoxylin for 5 minutes
2. Quickly rinse the slide in the beaker of water to remove the excess haematoxylin
3. Differentiate in 0.5% acetic acid for 30 seconds
4. Quickly rinse in the beaker of water
5. Place in Scott’s water (pH>7) until the sections “blue” (about 2 minutes)
6. Quickly rinse in water
7. Stain in alcoholic eosin for 5 minutes
8. Dehydrate section by dipping twice in 70% Ethanol. Do not wash in water or leave the
eosin in the alcohol (the eosin will wash out)
9. Place in 90% alcohol for 20 seconds
10. Place in absolute alcohol for 2 minutes
11. Place in ‘Histoclear’ - if the sections appear cloudy go back to step 9
12. Mount using a cover slip and DPX
Gomori rapid one step trichrome
Using the slide of rat kidney:
1. Stain in Harris Alum Haematoxylin for 5 minutes
2. Quickly rinse the slide in the beaker of water to remove the excess haematoxylin
3. Differentiate in 0.5% acetic acid for 30 seconds
4. Quickly rinse in the beaker of water
5. Place in Scott’s water (pH>7) until the sections “blue” (about 2 minutes)
6. Quickly rinse in water
7. Stain in Trichrome stain for 10 minutes
8. Quickly rinse the slide in the beaker of water to remove the excess Gomori
9. Two dips in 0.5% acetic acid
10. Quickly rinse in the beaker of water
Introduction:
Cellular components differ in their absorption of light and most tissue sections are invisible
using ordinary light microscopes - therefore, histological stains are used to treat the sections
that bind selectively to different structures according to their charges, making them different
colours. During this practical, the main aim was to visualise the muscle, lung and kidney rat
tissues using Hematoxylin and Eosin stain (H&E). Hematoxylin is a basic dye that binds to
negatively charged structures such as DNA and RNA due to negatively charged phosphate
groups in the sugar-phosphate backbone, and makes them appear violet under the microscope.
On the other hand, Eosin stain binds to positively charged substances such as amino acids and
proteins present in muscle fibres and cytoplasmic filaments, making them appear pink/red.
Hematoxylin and Trichrome stain was used to visualise the kidney tissue.
Materials and method:
Hematoxylin and Eosin (H&E): For lung and muscle tissues
1. Stain in Harris Alum Haematoxylin for 5 minutes
2. Quickly rinse the slide in the beaker of water to remove the excess haematoxylin
3. Differentiate in 0.5% acetic acid for 30 seconds
4. Quickly rinse in the beaker of water
5. Place in Scott’s water (pH>7) until the sections “blue” (about 2 minutes)
6. Quickly rinse in water
7. Stain in alcoholic eosin for 5 minutes
8. Dehydrate section by dipping twice in 70% Ethanol. Do not wash in water or leave the
eosin in the alcohol (the eosin will wash out)
9. Place in 90% alcohol for 20 seconds
10. Place in absolute alcohol for 2 minutes
11. Place in ‘Histoclear’ - if the sections appear cloudy go back to step 9
12. Mount using a cover slip and DPX
Gomori rapid one step trichrome
Using the slide of rat kidney:
1. Stain in Harris Alum Haematoxylin for 5 minutes
2. Quickly rinse the slide in the beaker of water to remove the excess haematoxylin
3. Differentiate in 0.5% acetic acid for 30 seconds
4. Quickly rinse in the beaker of water
5. Place in Scott’s water (pH>7) until the sections “blue” (about 2 minutes)
6. Quickly rinse in water
7. Stain in Trichrome stain for 10 minutes
8. Quickly rinse the slide in the beaker of water to remove the excess Gomori
9. Two dips in 0.5% acetic acid
10. Quickly rinse in the beaker of water
