STAINS FOR CARBOHYDRATE AND LIPID
PREPARED BY MR. ABHIJIT DAS
COMBINED PAS-ALCIAN BLUE
STAINING
Introduction:
This staining method is used to differentiate
between two types of carbohydrates: neutral carbohydrates (like glycogen) and
acidic carbohydrates (like mucins) in tissue samples.
Principle:
- PAS
Staining: PAS (Periodic Acid-Schiff)
highlights neutral carbohydrates.
Periodic acid reacts with these carbohydrates and then Schiff’s reagent
gives them a magenta (pink-purple)
color.
- Alcian
Blue Staining: Alcian Blue stains acidic carbohydrates blue.
It binds to acidic substances like mucins in tissues.
Procedure:
1.
Fixation:
Tissue samples are fixed in formalin or another mild fixative.
2.
Alcian Blue Staining:
The tissue is first stained with Alcian Blue, which makes acidic carbohydrates
appear blue.
3.
Periodic Acid:
Next, the tissue is treated with periodic acid, which prepares neutral
carbohydrates for staining.
4.
Schiff’s Reagent:
Schiff’s reagent is applied, which turns neutral carbohydrates magenta or
purple.
5.
Counterstaining (Optional):
A light background stain may be used to improve contrast.
Results:
- Acidic
carbohydrates: Stain blue.
- Neutral
carbohydrates: Stain magenta
or purple.
Applications:
- Medical
use: Helpful in diagnosing
diseases where carbohydrate content changes, such as certain cancers or
liver diseases.
- Research:
Used in studying connective tissues and
their carbohydrate content.
OIL RED O STAINING
Introduction:
Oil Red O staining is used to visualize fat
(neutral lipids) in tissue samples, especially in studies of fat-related
diseases like fatty liver.
Principle:
Oil Red O is a fat-soluble dye that binds to neutral
fats, such as triglycerides and lipoproteins, staining them red.
Procedure:
1.
Tissue Fixation:
The tissue is either frozen directly or fixed using a mild fixative like
formalin to keep the fats intact.
2.
Sectioning:
Thin slices of the frozen tissue (8–10 microns) are cut using a cryostat.
3.
Staining:
o The
tissue sections are placed in an Oil Red O solution, which stains the fat red.
4.
Rinsing:
The sections are rinsed in water to remove excess dye.
5.
Mounting:
The sections are mounted using a water-based medium because organic solvents
would dissolve the stained lipids.
Results:
- Lipids:
Neutral lipids, like triglycerides, stain bright
red.
- Other
tissue structures: Appear colorless or lightly
stained, often with a pale blue if counterstained with hematoxylin.
Applications:
- Clinical
Uses:
- Fatty
Liver: It helps detect fat accumulation
in the liver, often seen in diseases like non-alcoholic fatty liver
disease.
- Atherosclerosis:
Used to visualize fat deposits in arteries.
- Lipid
Storage Diseases: Identifies abnormal fat
storage in tissues.
- Research:
Frequently used in studies of obesity, lipid metabolism, and fat
distribution in the body.
Advantages:
- Specific
for Neutral Fats: Oil Red O selectively stains
neutral fats like triglycerides.
- Simple: The procedure is quick and easy to perform on frozen sections.
SUDAN BLACK B STAINING FOR
LIPIDS
Principle:
Sudan Black B is a fat-soluble dye that dissolves in lipids and stains them blue-black. It is useful for detecting neutral fats, phospholipids,
and cholesterol esters in tissues.
Procedure:
1.
Sectioning:
Frozen tissue sections are cut at 10 µm thickness.
2.
Fixation:
Sections are fixed in formalin.
3.
Staining:
Sections are placed in 0.5% Sudan Black B solution in
70% ethanol for 10-30
minutes.
4.
Rinsing:
Sections are rinsed in 70% ethanol to remove
excess dye.
5.
Counterstaining:
Hematoxylin is applied to stain the nuclei blue.
6.
Mounting:
A water-based medium is used to protect lipids.
Results:
- Lipids are stained blue-black.
- Nuclei appear blue
due to haematoxylin counterstaining.
- Non-lipid components remain unstained.
Applications:
- Fatty
liver disease: Shows fat deposits in liver tissue.
- Atherosclerosis:
Detects lipid-laden cells (cells or
tissues that are filled or loaded with lipids)
in arteries.
- Lipid
storage diseases: Shows abnormal lipid accumulation
in tissues.
Advantages:
Ø Specificity
for Lipids: Sudan Black B selectively stains lipids, allowing clear visualization of fat
deposits in tissues.
Ø Versatility:
It can stain a wide range of lipid types, including neutral
fats, phospholipids, and cholesterol esters, making it useful in various
diagnostic contexts.
Ø Diagnostic
Value: It is effective in diagnosing conditions like fatty liver disease, atherosclerosis
etc.
Ø Simple
and Cost-effective: The staining procedure is relatively simple
and inexpensive.
FERRIC HAEMATOXYLIN STAINING
FOR PHOSPHOLIPIDS
Introduction: Ferric
haematoxylin staining is used to detect phospholipids
in tissues, especially in nerve and myelin-rich areas.
Principle: Ferric ions oxidize haematoxylin, creating a complex that binds to phospholipids.
This makes phospholipids appear black under
the microscope.
Procedure:
1.
Fix tissue in formalin.
2.
Dehydrate the tissue in alcohol.
3.
Stain with ferric haematoxylin solution.
4.
Differentiate with an acidic solution to
remove excess stain.
5.
Mount the slides for observation.
Advantages:
- Specific for phospholipids.
- Clear, dark staining of lipid structures.
Disadvantages:
- Time-consuming.
- Requires
careful handling.